WO2021058145A1

WO2021058145A1 - Phage t7 promoters for boosting in vitro transcription

Info

Publication number: WO2021058145A1
Application number: PCT/EP2020/066070
Authority: WO
Inventors: Thomas Conrad; Sascha Sauer
Original assignee: Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft
Priority date: 2019-09-24
Filing date: 2020-06-10
Publication date: 2021-04-01

Abstract

The invention relates to a DNA polynucleotide, a kit comprising said DNA polynucleotide, and a method for in vitro transcribing (IVT) RNA using said DNA polynucleotide, wherein said DNA polynucleotide comprises (i) a T7 promoter sequence comprising (1) a T7 core promoter sequence and (2) a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising three guanines at positions +1 to +3, an adenine or thymine at position +4, at least two adenines at positions +4 to +8 and/or a thymine at position +4, and at most one cytosine at positions +4 to +8, and downstream thereof (ii) a nucleotide sequence encoding the RNA to be transcribed; and wherein the method comprises (a) providing said DNA polynucleotide as an IVT template, and (b) in vitro transcribing said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.

Description

Phage T7 promoters for boosting in vitro transcription

The present invention relates to a DNA polynucleotide and a method for in vitro transcribing (IVT) RNA using said DNA polynucleotide. In particular, the DNA polynucleotide comprises (i) a T7 promoter sequence comprising (1) a T7 core promoter sequence and (2) a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising three guanines at positions +1 to +3, an adenine or thymine at position +4, at least two adenines at positions +4 to +8 and/or a thymine at position +4, and at most one cytosine at positions +4 to +8, and downstream thereof (ii) a nucleotide sequence encoding the RNA to be transcribed. Furthermore, the method comprises (a) providing said DNA polynucleotide as an IVT template, and (b) in vitro transcribing said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.

Background

Obtaining a comprehensive understanding of RNA biology and the biology of diseases associated with RNA dysregulation is crucial as RNAs have been considered as the major determinant for converting genomic information from DNA into biological function. Thus, research has been focusing on the transcriptome of cells including RNAs that are translated into protein as well as regulatory RNAs. Unraveling RNA biology and the biology of diseases associated with RNA dysregulation requires detection, quantification, and characterization of different types of RNAs. However, RNAs exist with strongly varying copy numbers per cell and many approaches require a minimum amount of a given target RNA for further investigation. Hence, approaches are of interest for amplification of RNAs that maintain the relative abundances of different RNAs present in the original cell or tissue.

Different approaches exist for an efficient amplification of nucleic acids which is critical for many molecular biology procedures (Sauer et ah, Nat. Rev. Genet. 6, 465-476, 2005). A widely applied procedure being core to a host of analytical genomics applications is the in vitro transcription (IVT) of RNA using efficient viral promoter sequences for RNA amplification (Melton et al, Nucleic Acids Res. 12, 7035-7056, 1984). As examples, recent single-cell RNA- sequencing (scRNA-seq) methods such as CEL-seq (Hashimshony et al, Cell Rep. 2012 Sep 27;2(3):666-73), CEL-seq2 (Hashimshony et al, Genome Biol. 17, 77, 2016) and microdroplet- based procedures (Klein, A. M. et al. , Cell 161,1187-1201, 2015), rely on IVT using a T7 promoter and optimized reaction conditions (e.g. ionic strength and temperature) for engineered RNA polymerases. Moreover, IVT using a T7 promoter has been applied for low-amount DNA sequencing using linear amplification via transposon insertion (LIANTI) that uses the Tn5 transposon to fragment the genome and simultaneously insert a T7 RNA promoter for in vitro transcription (IVT) (Chen, C. et al, Science , 356, 189-194, 2017) and more recently sci-L3, a single-cell/nuclei sequencing method that combines combinatorial indexing (sci-) and linear (L) amplification (Yin, Y. et al., Mol Cell. 2019 Aug 28. pii: S1097-2765(19)30618-5, 2019, doi: 10.1016/j.molcel.2019.08.002. [Epub ahead of print]). Compared to exponential amplification by PCR, linear amplification by IVT features a much lower amplification bias (Hashimshony et al., Cell Rep. 2012 Sep 27;2(3):666-73). Further recent procedures comprising single-cell DNA sequencing such as scDam&T-seq (Rooijers (2019), Nat. Biotechnol. 37) or Chromatin Integration Labeling sequencing (CHIL-Seq; Harada (2019), Nat. Cell Biol. 21) also rely fundamentally on T7-promoter based in vitro transcription. IVT has also gained significant commercial interest for therapeutic or biotechnological applications. This includes the synthesis of RNA-based vaccines, mRNAs for transfection of cells for (over)expressing a protein without leaving a footprint in the genome, and regulatory RNAs for modulating gene or protein expression in vitro and in vivo ; Sahin et al.; Nat Rev Drug Discov. 2014 Oct;13(10):759-80. For example, CRISPRRNAs or (single) guide RNAs can be used for disrupting or restoring the sequence of target genes, and antisense RNAs and/or small- interfering RNAs can be used for translation inhibition of target mRNAs.

Many molecular biology procedures for an efficient amplification of nucleic acids take advantage of the phage T7 promoter. The T7 promoter refers to a 23 nucleotides long sequence comprising a transcription start site (TSS) denoted position +1. During transcription initiation, the T7 RNA polymerase binds the promoter DNA from nucleotide position -17 to -5 with high specificity, while the DNA double strand is melted from -4 to +2 to prime RNA synthesis from a GTP nucleotide at +1. Furthermore, it has been shown that sequences of the T7 core promoter, in particular the region from -17 to -1, and the first three transcribed nucleotides (+1 to +3) can affect the transcriptional activity, i.e. the promoter strength (defined by relative production of transcripts from a promoter; Ikeda, Biol. Chem. 267,11322-8, 1992) and that sequences upstream of the T7 core promoter positively affect T7 polymerase binding affinity to an IVT DNA template (Tang et al., Biol. Chem. 280,40707-13, 2005). However, it has been also suggested to only specify the +1 and +2 nucleotides as guanines (e.g. https://www.neb.com/protocols/2015/ll/24/sgrna-synthesis-using-the-hiscribe-quick-f7-high- yield-ma-synthesis-kit-neb-e2050; downloaded on 30.08.2019). Furthermore, to date, the sequence further downstream was not considered to be relevant for regulating the promoter strength; Patwardhan et al.; Nat Biotechnol. 2009 Dec;27(12): 1173-5. doi: 10.1038/nbt.1589. Moreover, a comprehensive characterization of the impact of flanking promoter sequences on transcription has not been shown. It is therefore unknown whether the extended downstream flanking sequence affects the T7 promoter strength or whether certain upstream and downstream flanking sequences affect the T7 promoter strength. However, there is still a need to expand the repertoire of effective T7 promoters. In particular, optimized T7 promoters with stronger outcome would be advantageous for a variety of applications such as linear amplification procedures.

Summary

The present application addresses the need for optimized T7 promoter sequences and methods using said promoter sequences for enhanced amplification of antisense RNA (aRNA) and/or improved in vitro transcription of RNAs by providing the embodiments as recited in the claims.

In particular, the present invention relates in preferred aspects to a DNA polynucleotide, a kit comprising said DNA polynucleotide, and a method for in vitro transcribing (IVT) RNA using said DNA polynucleotide. In particular, said DNA polynucleotide comprises (i) a T7 promoter sequence comprising (1) a T7 core promoter sequence and (2) a directly adjacent downstream flanking region, and downstream thereof (ii) a nucleotide sequence encoding the RNA to be transcribed.

The downstream flanking region of said DNA polynucleotide comprises eight bases (+1 to +8) comprising {three guanines at positions +1 to +3 (GGG)},

{an adenine (A) or thymine (T) at position +4},

{ [at least two adenines at positions +4 to +8] and/or [ a thymine at position +4] }, and {at most one cytosine (C) at positions +4 to +8}. Furthermore, the method comprises the steps of (a) providing said DNA polynucleotide as an IVT template, and (b) in vitro transcribing the RNA from said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates. The present invention relates in a further preferred aspect to a method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene, wherein the method comprises the steps of (a) reverse transcribing an RNA to the first strand of a cDNA comprising annealing a DNA polynucleotide according to the present invention, and (b) transcribing the cDNA into RNA using a T7 polymerase.

It has surprisingly been found that the +4 to +8 bases/nucleotides of the downstream flanking region of the T7 promoter drastically affect its transcription activity/efficiency/strength. As demonstrated herein, combinations of bases/nucleotides at the positions +4 to +8 according to defined rules described in the following have positive effects compared to a T7 core promoter with a conserved guanine-triplet (GGG) at positions +1 to +3 and a random sequence at positions +4 to +8. More specifically, it was found that at positions +4 to +8 sequences that are rich in adenines and thymines (AT -rich) improve the transcription strength of the T7 promoter, especially compared to respective sequences that are rich in guanines and cytosine (GC-rich). In particular, it was surprisingly found that T7 promoters, comprising

(an adenine or thymine at position +4},

{ [at least two adenines at positions +4 to +8] and/or

[a thymine at position +4], and

(at most one cytosine at positions +4 to +8} are advantageous for efficiently increasing transcription activity and thus, the efficiency of the T7 promoter. In addition, it was surprisingly found that the combination of a downstream flanking region according to the present invention with an AT-rich upstream flanking region further enhances the transcription activity of the T7 promoter. This is especially advantageous in applications wherein the in vitro transcription template is present and/or available at low concentration, e.g. a very low concentration as described herein.

An optimized T7 promoter according to the present invention can be readily applied in various IVT-based methods. It is especially advantageous when aiming at boosting linear amplification of nucleic acids. In particular, the T7 promoters of the invention comprising a defined +1 to +8 downstream flanking region ensure efficient amplification of target polynucleotides in a sample while avoiding a bias in IVT efficiency between different target polynucleotides. Optimized T7 promoters can be further used for increasing the efficiency of the synthesis of RNA in vitro , e.g. to produce large amounts of unmodified or modified RNA or recombinant proteins, preferably for therapeutic applications. An optimized T7 promoter is also highly valuable for IVT-based RNA-sequencing methods such as CEL-seq2, inDrop and MARS-seq as especially linear amplification of antisense RNA (aRNA) can be boosted and/or performed more accurately by using a reverse transcription primer comprising the optimized T7 promoter according to the invention instead of a commercially available reverse transcription primer. As illustrated in the appended examples, employing an inventive T7 promoter provided herein in a single cell RNA-seq method allows to detect more gene transcripts, i.e. mRNA of lowly expressed genes, for example of important cell state determinants such as transcription factors and chromatin modifiers. Moreover, many of those lowly expressed genes were only detected with a reverse transcription primer comprising the inventive T7 promoter but not with the conventional CEL-Seq2 reverse transcription primer. In addition, more transcripts per gene were detected in a single cell over the entire expression range compared to the conventional CEL-Seq2 reverse transcription primer. Thus, the expression level of a gene in a single cell can be determined more precisely, i.e. with a reduced variability. Thus, the sensitivity and/or accuracy of analytical methods used for detecting and/or sequencing nucleic acid molecules can be increased by employing an inventive T7 promoter provided herein, which is especially advantageous, for example, in case of analyzing transcriptomes on a single-cell level and/or detecting polynucleotides in liquid biopsies, i.e. in case raw material such as circulating tumor cells (CTC) is scarce and/or precious (Lawson et al., Nat. Cell Biol. 20,1349- 1360 (2018). As for RNA applications, an optimized T7 promoter is also highly valuable for IVT-based DNA- sequencing methods such as LIANTI, CHIL-Seq, Dam-seq (e.g. scDam&T-seq) or the sci-L3 method.

General terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

Detailed description

In a first aspect, the present invention relates to a DNA polynucleotide, comprising a T7 promoter comprising a T7 core promoter sequence and a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising {three guanines at positions +1 to +3},

{an adenine or thymine at position +4},

{ [at least two adenines at positions +4 to +8 and/or

[a thymine at position +4], and

{at most one cytosine at positions +4 to +8}.

Herein, pairs of brackets, i.e. “{ “[ ]” and “< >”, are used to specify relationships and layers within rules defined herein, with terms and expressions being grouped within an opening and a closing bracket of the same type, e.g. “{}”. Hence, brackets are to be understood as auxiliary means only for visually simplifying specific relationships and layers within rules defined herein.

In the context of the present invention, the term “polynucleotide” refers to a polynucleotide of at least 13 nucleotides covalently bonded in a chain and thus, representing a sequence of nucleotides. Herein, the term “nucleotide” refers to deoxyribonucleotides in case of DNA and cDNA (complementary DNA) molecules and to ribonucleotides in case of RNA molecules. Nucleotides consist of a nitrogenous base (also known as nucleobase), a five-carbon sugar (ribose in case of RNA or deoxyribose in case of DNA and cDNA), and at least one phosphate group. Nucleotides comprising as the respective nitrogenous base adenine, guanine, cytosine, thymine, and uracil are referred to as A, C, G, T and U nucleotides, respectively. Said nucleotides are preferred, though the term nucleotides can further comprise nucleotide analogues, such as peptide nucleic acids, morpholinos and/or locked nucleic acids, and/or modified nucleotides. Modified nucleotides include for example nucleotides comprising nucleobases that are methylated, formylated or hydroxylated nucleobases. Examples for modified or analogous nucleobases include for example 5-methyl-cytosine, 5-hydroxymethyl- cytosine, N6-methyladenine, 7-methylguanine, 2-aminopurine, pyrrolo-dC, 5-bromouracil, and hypoxanthine. Hence, the term “polynucleotide” refers to single- or double-stranded DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecule built up of A, C, G, T and/or U nucleotides and/or modified versions thereof. Different types of RNA molecules exist including, but not limited to mRNA, IncRNAs, circular RNAs, miRNAs, snoRNAs, tRNAs, snRNAs, siRNAs, crRNAs and sgRNAs.

In the context of the invention, the terms “adenine”, “cytosine”, “guanine”, “thymine” and “uracil” refer not only to the respective nucleobase, but also to the respective nucleotide or nucleoside. For example, an “adenine”, “A” or “adenosine” refers to an “adenine”, “adenosine”, “adenosine triphosphate” (“ATP”), “adenosine diphosphate” (“ADP”) or “adenosine monophosphate” (“AMP”).

The polynucleotide of the invention is a polynucleotide, preferably a DNA polynucleotide, more preferably a double-stranded DNA polynucleotide.

A single-stranded polynucleotide consists of one nucleotide sequence and a double-stranded polynucleotide of two complementary nucleotide sequences. The term “complementary” refers to nucleotides, the nitrogenous bases of which can naturally bind to each other by hydrogen bonds, i.e. A and T nucleotides, A and U nucleotides as well as C and G nucleotides. Both, a nucleotide and a nucleotide sequence have a 5’ (five prime) end and a 3’ (three prime) end based on the five carbon sites of sugar molecules in adjacent nucleotides. A nucleotide sequence is read herein from its respective 5’ to its respective 3’end, wherein the term “downstream” refers to a nucleotide which is in 3’ direction of a reference nucleotide. The term “upstream” refers to a nucleotide which is in 5’ direction of a reference nucleotide within a respective nucleotide sequence. A nucleotide which is located downstream of a reference nucleotide within a respective nucleotide sequence, can be directly, i.e. covalently, linked to the 3’ end of said reference nucleotide, which is referred to herein as “directly adjacent”. A nucleotide which is located upstream of a reference nucleotide within a respective nucleotide sequence, can be directly, i.e. covalently linked to the 5’ end of said reference nucleotide, which is also referred to herein as “directly adjacent”. The polynucleotide, preferably the DNA polynucleotide, according to the present invention comprises a T7 promoter sequence comprising a T7 core promoter sequence and a directly adjacent downstream flanking region. The terms “promoter” and “promoter sequence” are used interchangeably herein and refer to a nucleotide sequence required for initiation of transcription of a target nucleotide sequence by DNA-binding enzymes such as RNA polymerase, transcription factors and/or epigenetic modifiers, e.g. histone acetylases, histone methylases or DNA-methylases. As the promoter is a T7 bacteriophage (Escherichia virus T7) promoter, the respective enzyme(s) binding to it preferably comprise a T7 RNA polymerase. The T7 promoter and the T7 RNA polymerase, as used herein, are derived from a T7 bacteriophage and thus, may comprise the sequence of the naturally occurring T7 bacteriophage core promoter (e.g. SEQ ID NO:l) and RNA polymerase, respectively, and/or may comprise modifications compared to their respective naturally occurring sequence. The T7 RNA polymerase may also be a recombinant protein and/or comprise altered post-translational modifications.

Herein, the term “T7 promoter” is used interchangeably with the term “T7 promoter sequence” and refers to a nucleotide sequence comprising a transcription start site (TSS) which is denoted as position +1. Positions downstream the TSS are denoted herein with a positive sign (“+”) and positions upstream of the TSS with a negative sign (“-”). The T7 promoter sequence comprises at least a T7 core promoter sequence and a directly adjacent downstream flanking region that is directly, i.e. covalently, linked to the 3’ end of said T7 core promoter sequence.

The term “T7 core promoter sequence”, herein also referred to as “T7 core promoter”, refers to the nucleotide sequence upstream of the TSS, more specifically to the nucleotide sequence from positions -1 to -17 and has the nucleotide sequence TAATACGACTCACTATA (SEQ ID NO:l).

Herein, the downstream flanking region comprises eight positions (positions +1 to +8 of the T7 promoter sequence) that have a nucleotide sequence composed according to certain rules. In particular, the downstream flanking region comprises

(three guanines at positions +1 to +3},

(an adenine or thymine at position +4},

{ [at least two adenines at positions +4 to +8] and/or

[a thymine at position +4] }, and

(at most one cytosine at positions +4 to +8}. Thus, the TSS is preferably a G nucleotide. Furthermore, also the two nucleotides at positions +2 and +3 are preferably G nucleotides.

Surprisingly, it was found that the number and concrete position of certain nucleotides at positions +4 to +8 influence the activity of the T7 promoter. In particular, it was found that at least three adenines or thymines at positions +4 to +8 are advantageous for enhancing the activity of the T7 promoter. It was further found that, in particular, the nucleotides at positions +4 to +7 and especially at position +4 have a strong impact on the performance of the T7 promoter. The absence of a cytosine is preferred within positions +4 to +7, in particular within positions +4 to +6 and/or in particular when the cytosine directly follows a thymine and/or directly precedes a guanine. It was further found that sequences of covalently linked thymines (e.g. TT, TTT or TTTT) have a negative effect on the T7 promoter strength, in particular when three or more consecutive thymines are present.

Furthermore, it was surprisingly found that the combination of rules can result in an increased effect compared to sequences according to only one of the respective rules. For example, a negative effect of sequences of covalently linked thymines on T7 promoter activity is reduced in the absence of a guanine or cytosine at positions +4 to +8 or the presence of an adenine at position +4. As another example, a negative effect of a cytosine within positions +4 to +6 on T7 promoter activity is reduced when at least three adenines are present at positions +4 to +8. As a further example, a guanine at position +5 has a positive effect on T7 promoter activity in combination with an adenine or thymine at position +4 and/or an adenine at position +6. Hence, GGGAGA and GGGTGA at positions +1 to +6 can have positive effects on T7 promoter activity despite the presence of a guanine within positions +4 to +8.

In certain embodiments, the downstream flanking region further comprises at least two adenines at positions +4 to +8.

In certain embodiments, the downstream flanking region further comprises

{ [at least three adenines or thymines at positions +4 to +8] and/or

[a guanine followed by an adenine within positions +5 and +6] }.

In certain embodiments, the downstream flanking region does not comprise three consecutive thymines.

In certain embodiments, the downstream flanking region does not comprise three consecutive thymines within positions +4 to +8. In certain embodiments, the downstream flanking region does not comprise

{two consecutive thymines within positions +4 and +7, when

[a cytosine or guanine is present at positions +4 to +8], or

[a thymine, cytosine or guanine is present at position +4] } .

In certain embodiments, the downstream flanking region does not comprise two consecutive thymines within positions +4 and +7.

In certain embodiments, the downstream flanking region does not comprise a thymine followed by a cytosine within positions +4 and +7.

In certain embodiments, the downstream flanking region does not comprise a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8.

In certain embodiments, the downstream flanking region does not comprise a cytosine within positions +4 to +6.

In certain embodiments, the downstream flanking region does not comprise a cytosine followed by a guanine within positions +4 to +7.

In preferred embodiments, the downstream flanking region comprises { [at least three adenines or thymines at positions +4 to +8] and/or

[a guanine followed by an adenine within positions +5 and +6] }, and said downstream flanking region does not comprise { [three consecutive thymines within positions +4 to +8], and/or [two consecutive thymines within positions +4 and +7 when

<a cytosine or guanine is present at positions +4 to +8> or <a thymine, cytosine or guanine is present at position +4> ] },

{a thymine followed by a cytosine within positions +4 and +7},

{a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8, and

{a cytosine followed by a guanine within positions +4 to +7}. In certain embodiments, the downstream flanking region further comprises an adenine at position +4.

In certain embodiments, the downstream flanking region has the sequence GGGAGAGT.

In certain embodiments, the downstream flanking region further comprises at most one guanine or cytosine at positions +4 to +8.

It was surprisingly found that an adenine at position +4 improves the T7 promoter strength compared to any other nucleotide at this position. It was further found, that downstream sequences are advantageous that do not comprise a guanine within positions +4 to +8 except in the case the nucleotide is covalently linked to an adenine in the context of an AGA (or TGA) nucleotide sequence within positions +4 to +6. Thus, more than one guanine or cytosine within positions +4 to +8 is preferably avoided for increasing the activity of the T7 promoter.

Hence, in preferred embodiments, the downstream flanking region comprises

{ [at least three adenines or thymines at positions +4 to +8] and/or [a guanine followed by an adenine within positions +5 and +6], and said downstream flanking region does not comprise

{ [three consecutive thymines within positions +4 to +8], and/or

[two consecutive thymines within positions +4 and +7 when

(a thymine followed by a cytosine within positions +4 and +7},

(a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8}, and

(a cytosine followed by a guanine within positions +4 to +7}; wherein the downstream flanking region further comprises (an adenine at position +4} and (at most one guanine or cytosine at positions +4 to +8}. Preferably, the downstream flanking region is selected from the group consisting of GGGAAATA, GGGAAAAT, GGGAATAT, GGGATAAT, GGGAGAAT, GGGAATAC, GGGAAGTA, GGGAGATT, GGGAGATA, GGGAAATG, GGGAAAAC and GGGAAAGT.

It was further surprisingly found that position +8 has the smallest effect on the T7 promoter activity among positions +4 to +8.

Thus, in certain embodiments, the downstream flanking region is selected from the group consisting of GGGAAATN, GGGAAAAN, GGGAATAN, GGGATAAN, GGGAGAAN, GGGAATAN, GGGAAGTN, GGGAGATN, GGGAGATN, GGGAAATN, GGGAAAAN and GGGAAAGN; wherein N denotes A, C, G, T or U.

In other preferred embodiments,

{the downstream flanking region comprises three guanines at positions +1 to +3},

{an adenine at position +4},

{at least two adenines at positions +4 to +8}, and { [at least three adenines or thymines at positions +4 to +8] and/or

[a guanine followed by an adenine within positions +5 and +6]; and does not comprise

{more than one guanine or cytosine at positions +4 to +8},

{ [three consecutive thymines within positions +4 to +8] and/or [two consecutive thymines within positions +4 and +7, when

<a cytosine or guanine is present at positions +4 to +8> or

<a thymine, cytosine or guanine is present at position +4> ] },

{a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8}, {and a cytosine followed by a guanine within positions +4 to +7}.

In other preferred embodiments, the downstream flanking region comprises {three guanines at positions +1 to +3},

{an adenine at position +4},

{ [at least three adenines at positions +4 to +8] and/or

[a guanine followed by an adenine within positions +5 and +6] },

{at most one guanine or cytosine at positions +4 to +8},

{at most two consecutive thymines within positions +4 and +8},

{no consecutive thymines within positions +4 and +7} and {no cytosine within positions +4 to +6}.

In certain embodiments, the downstream flanking region does not comprise a thymine at position +5 followed by a guanine at position +6.

In certain embodiments, the downstream flanking region does not comprise two consecutive thymines within positions +4 to +8.

In certain embodiments, the downstream flanking region does not comprise {a thymine at position +5 followed by a guanine at position +6} and {two consecutive thymines within positions +4 to +8}.

It was further surprisingly found that already two consecutive thymines within positions +4 to +8 might have a slightly negative effect on the activity of the T7 promoter in some nucleotide sequence compositions and that also a thymine-guanine doublet (TG) at positions +5 and +6 is preferably avoided for increasing T7 promoter activity.

Hence, in preferred embodiments, the downstream flanking region comprises

{ [at least three adenines or thymines at positions +4 to +8 and/or

[a guanine followed by an adenine within positions +5 and +6], and said downstream flanking region does not comprise

{ [three consecutive thymines within positions +4 to +8], and/or

[two consecutive thymines within positions +4 and +7 when <a cytosine or guanine is present at positions +4 to +8> or

<a thymine, cytosine or guanine is present at position +4> ] },

{a thymine followed by a cytosine within positions +4 and +7},

{a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8},

{a cytosine followed by a guanine within positions +4 to +7}; wherein the downstream flanking region further comprises

{an adenine at position +4 and at most one guanine or cytosine at positions +4 to +8}; and wherein the downstream flanking region does not comprise {a thymine at position +5 followed by a guanine at position +6}, and

{two consecutive thymines within positions +4 to +8}.

Preferably, the downstream flanking region is selected from the group consisting of GGGAAATA, GGGAAAAT, GGGAATAT, GGGATAAT, GGGAGAAT, GGGAATAC and GGGAAGTA. In certain embodiments, the downstream flanking region is selected from the group consisting of GGGAAATN, GGGAAAAN, GGGAATAN, GGGATAAN, GGGAGAAN, GGGAATAN and GGGAAGTN; wherein N denotes A, C, G, T or U.

In other preferred embodiments, the downstream flanking region comprises {three guanines at positions +1 to +3 },

{an adenine at position +4},

{more than one guanine or cytosine at positions +4 to +8},

{two consecutive thymines within positions +4 to +8}, (a thymine at position +5 followed by a guanine at position +6}, and

(a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8}. In further preferred embodiments, the downstream flanking region comprises (three guanines at positions +1 to +3},

(an adenine at position +4},

{ [at least three adenines at positions +4 to +8] and/or

[a guanine followed by an adenine within positions +5 and +6], (at most one guanine or cytosine at positions +4 to +8},

(no consecutive thymines within positions +4 and +8},

(no thymine at position +5 followed by a guanine at position +6}, and (no cytosine within positions +4 to +6}.

In certain embodiments, the downstream flanking region further comprises an adenine at position +4, three to four adenines at positions +4 to +8, and no guanines or cytosines at positions +4 to +8.

It was further surprisingly found that the complete absence of guanines and cytosines and consecutive thymines within positions +4 to +8 has a particularly strong positive effect on the transcription activity of the T7 promoter. It was further surprising that the presence of one or two thymines at positions +4 to +8 is preferred over the presence of only adenines.

Hence, in preferred embodiments, the downstream flanking region comprises

{ [at least three adenines or thymines at positions +4 to +8] and/or

{ [three consecutive thymines within positions +4 to +8], and/or

[two consecutive thymines within positions +4 and +7 when <a cytosine or guanine is present at positions +4 to +8> or <a thymine, cytosine or guanine is present at position +4> ] },

{a thymine followed by a cytosine within positions +4 and +7},

{a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8}, and

{a cytosine followed by a guanine within positions +4 to +7}; wherein the downstream flanking region further comprises {an adenine at position +4} and

{at most one guanine or cytosine at positions +4 to +8}; wherein the downstream flanking region does not comprise

{a thymine at position +5 followed by a guanine at position +6} and

{two consecutive thymines within positions +4 to +8}; wherein the downstream flanking region further comprises

{an adenine at position +4},

{three to four adenines at positions +4 to +8}, and {no guanines or cytosines at positions +4 to +8}, preferably wherein the downstream flanking region has the sequence GGGATAAT.

In particularly preferred embodiments, the downstream flanking region has a sequence selected from the group consisting of GGGADDDN, wherein D denotes A, G or T/U, and wherein N denotes A, C, G, T or U. In even more preferred embodiments, the sequence of the downstream flanking region is selected from the group consisting of GGGADDWH, preferably from GGGAWWWW, wherein D denotes an A, G or T/U, wherein W denotes A or T/U, and wherein H denotes A, C or T/U.

In further preferred embodiments, the downstream flanking region comprises three guanines at positions +1 to +3, an adenine at position +4, three to four adenines at positions +4 to +8, no guanine or cytosine at positions +4 to +8, and no consecutive thymines within positions +4 and +8.

In particularly preferred embodiments, the downstream flanking region has the sequence GGGAAATA (see the T7 promoter sequence as set forth in SEQ ID NO:21), GGGAAAAT (see the T7 promoter sequence as set forth in SEQ ID NO:23), GGAATAT (see the T7 promoter sequence as set forth in SEQ ID NO:25), GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17), or GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19). Preferably the downstream flanking region has the sequence GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17) or GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19), more preferably GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17).

In certain embodiments, the downstream flanking region is selected from the group consisting of GGGAAATN, GGGAAAAN, GGAATAN, GGGATAAN, or GGGAGAGN; wherein N denotes A, C, G, T or U.

Furthermore, when the DNA polynucleotide of the invention is used as a primer, i.e. a reverse transcription primer, e.g. for RNA sequencing, the length of the primer may be as small as possible. In particular, if the T7 promoter of the invention is used for improving a pre-existing protocol, it may be preferable to modify the pre-existing protocol as little as possible while profiting from the enhanced T7 promoter activity according to the invention. For example, for improving a CEL-Seq2 based protocol by replacing the T7 promoter, the downstream flanking region of the inventive T7 promoter may overlap with the sequencing adapter further downstream thereof. In other words, the pre-existing sequencing adapter may be part of the downstream flanking region, i.e. at positions +6 to +8 or +7 to +8.

Thus, in certain embodiments, the downstream flanking region further comprises the sequence AGT at positions +6 to +8, or the sequence AG at positions +7 to +8, preferably AGT at positions +6 to +8. In particular, this rule may be combined with other rules for the composition of the downstream flanking region, as provided herein, as far as there is no conflict. T7 promoters with such a downstream flanking region (i.e. with AGT at positions +6 to +8) may be particularly useful in some embodiments of the inventive sequencing methods provided herein. Thus, in one embodiment, the downstream flanking region has the sequence GGGAAAGT, GGGTAAGT, GGGATAGT, GGGTGAGT, or GGGAGAGT, preferably GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19).

In one further embodiment, the downstream flanking region has the sequence GGGAAAAG, GGGAATAG, GGGATAAG, GGGTGAAG, GGGTAAAG, GGGAGAAG, GGGTGTAG, GGGAGTAG, GGGATTAG.

Since a high-ranking 5’RACE-seq position (see Table 1) indicates a strong T7 promoter activity, and overlap with the CEL-Seq2 sequencing adapter downstream of the T7 promoter may be advantageous for certain sequencing applications, both a very high rank in Table 1, and a high rank in combination with an overlap with said sequencing adapter, may be particularly useful for said applications.

Thus, in one embodiment, the downstream flanking region has the sequence GGGAAATA (see the T7 promoter sequence as set forth in SEQ ID NO:21), GGGAAAAT (see the T7 promoter sequence as set forth in SEQ ID NO:23), GGAATAT (see the T7 promoter sequence as set forth in SEQ ID NO:25), GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17), GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19), GGGAAAGT, GGGTAAGT, GGGATAGT or GGGTGAGT, preferably GGGAGAGT.

However, in certain embodiments, the T7 promoter of the invention is selected primarily based on the 5’RACE-seq rank (Table 1).

Thus, in certain embodiments, the sequence of the downstream flanking region is selected from the group consisting of the “Pos. +1 to +8” sequences in Table 1 of ranks 1 to 510, ranks 1 to 176, ranks 1 to 132 or ranks 1 to 66, preferably ranks 1 to 66.

Thus, in certain embodiments, the sequence of the downstream flanking region is selected from the group consisting of the “Pos. +1 to +8” sequences in Table 1 of ranks 1 to 64, ranks 1 to 48, ranks 1 to 32, ranks 1 to 24, or ranks 1 to 12.

Moreover, in certain embodiments the sequence of the downstream flanking region does not have the sequence GGGGTTCA, GGGAGTTC or GGGATACC. In some embodiments, the sequence of the downstream flanking region does not have the sequence GGGTAGAT.

It was furthermore surprisingly found that certain nucleotide sequences comprising AATT, in particular GAATT, directly adjacent upstream of the T7 core promoter, further enhance the T7 promoter strength, especially in combination with a downstream flanking region according to the present invention. Further increasing the strength of an optimized T7 promoter sequence comprising a downstream flanking region as shown above by combining it with an upstream flanking region according to the present invention is particularly advantageous for approaches based on scarce and/or precious raw material (e.g. liquid biopsies) such as in case of in vitro transcription (IVT) when the concentration of the IVT template is low, e.g. very low as described herein, and/or analyses are performed, for example, on a single-cell level, or on less than 100 cells, preferably less than 10 cells, very preferably a single cell.

Herein, the term “upstream flanking region” refers to the sequence directly adjacent upstream of the T7 core promoter sequence and thus, covalently bound to its 5’ end. The upstream flanking region comprises 4 or 5 nucleotides, preferably 5 nucleotides.

In preferred embodiments, the upstream flanking region has the sequence AATT, GAATT, GATTT or GAAAT, preferably AATT or GAATT, even more preferably GAATT (see e.g. the T7 promoter sequences as set forth in SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26).

In some embodiments, the T7 promoter further comprises four bases consisting of adenines and/or thymines directly upstream of the T7 core promoter. Preferably, it further comprises a guanine directly upstream of said adenines and/or thymines.

In some embodiments, the T7 promoter comprises a downstream flanking region of the invention and an upstream flanking region of the invention.

In preferred embodiments, the T7 promoter further comprises the sequence AATT directly upstream of the T7 core promoter sequence.

In particularly preferred embodiments, the T7 promoter further comprises the sequence GAATT directly upstream of the T7 core promoter sequence.

The DNA polynucleotide according to the present invention can further comprise downstream of the T7 core promoter sequence a nucleotide sequence encoding an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPRRNA. In the context of the present invention, the terms “encoding” or “encode” refer to genetic information comprised in a DNA sequence that can be transcribed into an RNA molecule, i.e. that can be used as a template for synthesis of an RNA molecule.

In a double-stranded DNA polynucleotide, the term “nucleotide sequence” preferably refers to the strand that comprises the coding strand. As used herein, a coding strand comprises a sequence identical to the RNA it encodes. As used herein, a sequence is identical to another sequence if each nucleotide at each position is the same, except that a thymine can be in place of an uracil and an uracil can be in place of a thymine according to the nucleotides comprised in DNA and RNA, respectively.

The encoded RNA can be an in-vitro transcribed RNA according to the invention, in particular an mRNA or a non-coding RNA.

Herein, the terms “mRNA molecule” and “mRNA” are used interchangeably and refer to a class of RNA molecules, preferably single-stranded sequences built up of A, C, G, and U nucleotides that contain one or more coding sequences. Said one or more coding sequences can be used for example as a translation template during synthesis of an amino acid sequence and thus, for converting genomic information stored in an mRNA molecule into an amino acid sequence. In other words, the term “mRNA” should be understood to mean any RNA molecule which is suitable for the expression of an amino acid sequence or which is translatable into an amino acid sequence such as a protein.

More specifically, an mRNA comprises preferably a 5’ UTR, a coding sequence and a 3’ UTR. The 5’ end of the 5’ UTR is defined by the TSS and its 3’ end is followed by the coding sequence. The coding sequence is delineated by the start and the stop codon, i.e. the first and the last three nucleotides of the mRNA that can be translated, respectively. The 3’ UTR starts after the stop codon of the coding sequence and can be followed by a poly A tail.

The 5’ UTR generally comprises at least one ribosomal binding site (RBS) such as the Shine- Dalgarno sequence in prokaryotes and the Kozak sequence or translation initiation site in eukaryotes. RBS promote efficient and accurate translation of an mRNA molecule by recruiting ribosomes during initiation of translation. The activity of a given RBS can be optimized by varying its length and sequence as well as its distance to the start codon. Alternatively or optionally, the 5’ UTR comprises internal ribosome entry sites or IRES. Furthermore, a 5’ UTR can comprise one or more additional regulatory sequences such as a binding site for an amino acid sequence that enhances the stability of the mRNA, a binding site for an amino acid sequence that enhances the translation of the mRNA, a regulatory element such as a riboswitch, a binding site for a regulatory RNA molecule such as an miRNA, and/or a nucleotide sequence that positively affects initiation of translation. Furthermore, within the 5’ UTR there are preferably no functional upstream open reading frames, out-of-frame upstream translation initiation sites, out-of-frame upstream start codons and/or nucleotide sequences giving rise to a secondary structure that reduces or prevents translation. The presence of such nucleotide sequences in the 5’ UTR can have a negative effect on translation.

The mRNA may optionally comprise a 3’ UTR. The 3’ UTR may comprise one or more regulatory sequences such as a binding site for an amino acid sequence that enhances the stability of the mRNA molecule, a binding site for a regulatory RNA molecule such as a miRNA, and/or a signal sequence involved in intracellular transport of the mRNA.

The coding sequence of an mRNA comprises codons that can be translated into an amino acid sequence. The coding sequence can contain the codons of a naturally occurring coding sequence or it can be a partially or completely synthetic coding sequence. Alternatively, the coding sequence can be a partly or fully codon optimized sequence derived from the natural sequence to be used. Most of the amino acids are encoded by more than one codon, i.e. three consecutive nucleotides of an mRNA that can be translated into an amino acid. Codons exist that are used preferentially in some species for a given amino acid. The presence of more often occurring codons can enhance the amount of amino acid sequences translated based on a given mRNA molecule compared to the same mRNA molecule but comprising comparably rare codons. Hence, it is advantageous to species specific adapt the codons in a given coding sequence by avoiding rare codons and enhancing the occurrence of abundant codons for a given amino acid to improve the translation of said mRNA.

As regards the function of the encoded amino acid sequence, there is no limitation and possible amino acid sequences to be encoded by said mRNA. Herein, the term “amino acid sequence” encompasses any kind of amino acid sequence, i.e. chains of two or more amino acids which are each linked via peptide bonds and refers to any amino acid sequence of interest. Preferably, the encoded amino acid sequence is at least 5 amino acids long, more preferably at least 10 amino acids, even more preferably at least 50, 100, 200 or 500 amino acids. In other words, the term “amino acid sequence” covers short peptides, oligopeptides, polypeptides, fusion proteins, proteins as well as fragments thereof, such as parts of known proteins, preferably functional parts. These can, for example be biologically active parts of a protein or antigenic parts such as epitopes which can be effective in raising antibodies. Preferably, the function of the encoded amino acid sequence in the cell or in the vicinity of the cell is needed or beneficial, e.g. an amino acid sequence the lack or defective form of which is a trigger for a disease or an illness, the provision of which can moderate or prevent a disease or an illness, or an amino acid sequence which can promote a process which is beneficial for the body, in a cell or its vicinity. The encoded amino acid sequence can be the complete amino acid sequence or a functional variant thereof. Further, the encoded amino acid sequence can act as a factor, inducer, regulator, stimulator or enzyme, or a functional fragment thereof, where this amino acid sequence is one whose function is necessary in order to remedy a disorder, in particular a metabolic disorder or in order to initiate processes in vivo such as the formation of new blood vessels, tissues, etc. Here, functional variant is understood to mean a fragment which in the cell can undertake the function of the amino acid sequence whose function in the cell is needed or the lack or defective form whereof is pathogenic. Preferably, such an amino acid sequence is advantageous with respect to applications in supplemental or medical purposes to generate or regenerate physiological functions caused by suboptimal amino acid sequence biosynthesis and thus also to favorably influence directly or indirectly the course of diseases.

A preferred coding sequence downstream of the T7 core promoter encodes a transposase or a genome editing enzyme.

A transposase refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. Preferred transposases include Sleeping Beauty transposases and PiggyBac transposases.

A genome editing enzyme, as used herein, refers to an enzyme which recognizes a specific nucleotide sequence, preferably a DNA sequence, and cuts the nucleotide sequence at or nearby the recognition position. Some genome editing enzymes, in particular CRISPR associated proteins, such as, but not limited to Cas9, require a polynucleotide, for example, a CRISPR RNA or guide RNA, which guides them to the specific target sequence. Some genome editing enzymes, in particular ZNFs and TALENs recognize the specific target sequence by themselves.

Preferred genome editing enzymes include zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and CRISPR-associated proteins such as Cas9, Casl2b or Cpfl, preferably Cas9. A CRISPR-associated protein is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can target specific desired DNA sequences, which enables zinc-finger nucleases to target unique sequences even within complex genomes. The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs. The non-specific cleavage domain from the type IIs restriction endonuclease Fokl is typically used as the cleavage domain in ZFNs. Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA- binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The DNA binding domain contains a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition.

Genetic information comprised in a DNA sequence that can also be transcribed into non-mRNA molecules such as non-coding RNA molecules. Herein, the term “non-coding RNA” refers preferably to regulatory RNA such as microRNA (miRNA), small-interfering RNA (siRNA), antisense RNA, CRISPR RNA (crRNA), single guide RNA (sgRNA), guide RNA (gRNA), antagoNAT, or a precursor thereof such as pri-miRNA, pre-miRNA or short-hairpin RNA (shRNA).

A microRNA refers to a small non-coding RNA molecule (comprising about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules cleaved, destabilized by shortening the polyA tail of the mRNA and/or less efficiently translated into proteins by ribosomes. Hence, microRNA molecules can reduce the amount of protein produced by a respective mRNA.

Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA refer to double-stranded RNA molecules of 20 to 25 nucleotides in length. siRNA molecules are involved in the degrading mRNA after transcription, thus preventing their translation into proteins. An antisense RNA, as used herein, refers to a single stranded RNA that is complementary to another polynucleotide. Preferably, an antisense RNA is a non-coding RNA. For example, an antisense RNA can hybridize to the complementary polynucleotide. Preferably, the complementary polynucleotide is an RNA, preferably an mRNA. In preferred embodiments, the antisense RNA is used for inhibiting the translation of a complementary mRNA into protein by hybridization of the two RNAs. Preferably, the inhibition occurs within a living cell.

In other preferred embodiments, the antisense RNA is used for detecting a further polynucleotide by hybridization of the RNA to the polynucleotide. Preferably, the polynucleotide to be detected is an RNA, preferably an mRNA. The hybridization can occur outside or within a cell. Preferably, said cell is fixed. An antisense RNA which is used for detecting another polynucleotide is also termed “probe”.

In preferred embodiments, the antisense RNA is an amplified antisense RNA which is generated in the linear amplification method according to the invention. The amplified antisense RNA (aRNA) may be transcribed from a double-stranded DNA. Preferably, the amplified antisense RNA is transcribed from a cDNA. Preferably, the cDNA has been reverse-transcribed from an RNA, preferably an mRNA, by using the DNA polynucleotide of the invention as reverse transcription primer.

As used herein, the term “aRNA” refers to an amplified antisense RNA. An antagoNAT refers to a single stranded oligonucleotide which can inhibit a naturally occurring antisense RNA and thus, can be used for increasing gene expression, in particular by antagonizing said antisense RNA.

Herein, the term “CRISPR” refers to a “clustered regularly interspaced short palindromic repeats” sequence, and in particular to a CRISPR RNA (crRNA) and a single-guide RNA (sgRNA). A crRNA comprises a guide RNA which is an approximately 20 nucleotides long sequence and which guides genome editing enzymes such as CRISPR associated proteins to the respective nucleotide sequence to be edited which is preferably comprised in a double-stranded genomic DNA molecule. Some CRISPR associated proteins further require a trans-activating CRISPR RNA (tracrRNA), which can be provided as a single guide RNA (sgRNA) and thus a fusion product comprising at least one crRNA and a trans-activating CRISPR RNA (tracrRNA). The DNA polynucleotide according to the present invention can comprise downstream of the T7 core promoter sequence a nucleotide sequence encoding an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPR RNA, wherein the said nucleotide sequence is not, partially, or completely overlapping with the downstream flanking region directly adjacent to the T7 core promoter sequence. Preferably, said nucleotide sequence is not or partially overlapping with the downstream flanking region directly adjacent to the T7 core promoter sequence.

The DNA polynucleotide according to the present invention can further comprise a polyT- stretch, wherein the polyT-stretch is a nucleotide sequence of 9 to 35 consecutive thymines, preferably 22 to 27 consecutive thymines, more preferably 24 consecutive thymines, preferably followed by an adenine, cytosine or guanine, preferably at the 3’ end of the DNA polynucleotide.

Hence, the term “polyT-stretch” refers to a nucleotide sequence consisting of consecutive, i.e. covalently linked thymines. Said polyT-stretch is especially advantageous for using the DNA polynucleotide as a reverse transcription primer for reverse transcription of an RNA, e.g. an mRNA, into a cDNA. A “primer”, as used herein, refers to a poly- or oligonucleotide which comprises a sequence complementary to a sequence comprised in another poly- or oligonucleotide. Hence, a primer can be used for example for PCR, reverse transcription and/or the synthesis of the complementary strand of a DNA molecule.

In some embodiments, the DNA polynucleotide according to the present invention comprises downstream of the T7 core promoter sequence a polyT-stretch. Embodiments comprising the DNA polynucleotide and a polyT-stretch are especially advantageous for use as a reverse transcription primer.

The DNA polynucleotide according to the present invention can further comprise a polyT, and a sequencing adapter, wherein the sequencing adapter is a nucleotide sequence comprising a nucleotide sequence comprised in a library preparation primer, wherein the library preparation primer comprises a flow cell binding sequence, which is complementary to an oligo- or polynucleotide present at the surface of the flow cell of a next-generation-sequencing device, and wherein the sequencing adapter does not comprise more than eight consecutive thymines and/or only thymines.

A sequencing adapter, as used herein, refers to a polynucleotide which functions as a binding sequence for a polymerase chain reaction (PCR) primer and/or library preparation primer. In the context of the present invention, a library preparation primer (LPP) comprises a flow cell binding sequence (FCBS) and a sequence which is identical or highly similar to a sequence comprised in the sequencing adapter, such that the LPP can bind to the complementary sequence of the sequencing adapter. A flow cell binding site, as used herein, refers to an oligo- or polynucleotide which binds to a complementary sequence immobilized at the surface of a flow cell. A flow cell is a part of a sequencing device that can be loaded with the material to be sequenced, wherein a sequencing device refers to a device for determining the sequence of a polynucleotide. Preferably, the sequencing adapters are sequencing adapters used in next- generation sequencing methods comprising library preparation methods and in particular Illumina library preparation protocols. Hence, a preferred sequencing adapter sequence is a sequence comprised in an Illumina library preparation primer such as PEI, PE2 or PE2-N6 and suitable flow cell binding sequences are for example comprised in Illumina P5 and P7 oligo- or polynucleotides.

As regards an oligonucleotide as used herein, the same applies as it has been described in the context of a polynucleotide, except its length. Whereas a polynucleotide can comprise more than two, preferably 24 or more, or even up to 250 nucleotides, an oligonucleotide is to be understood as a sequence of two to 500, preferably 2 to 250 covalently linked nucleotides. Moreover, also the other features of such an oligonucleotide can be as described above.

In some embodiments, the DNA polynucleotide according to the present invention comprises a T7 promoter sequence and a sequencing adapter. This is especially advantageous for use of the DNA polynucleotide as a reverse transcription primer in approaches that comprise a sequencing step following reverse transcription of a nucleotide sequence encoding for example an mRNA, sgRNA, guide RNA or CRISPR RNA. The sequencing adapter can optionally at least partially overlap with the T7 promoter sequence and/or the polyT-stretch. Preferably, the sequencing adapter is directly adjacent downstream of the T7 promoter sequence.

In some embodiments, the DNA polynucleotide according to the present invention further comprises a barcode and/or a unique molecular identifier (UMI). A barcode, as used herein, refers to an oligonucleotide which can be used to uniquely label e.g. an RNA of a specific sample, in particular of one specific cell. An UMI refers herein to an oligonucleotide which can be used to specifically label one specific RNA molecule.

In particularly preferred embodiments of the present invention, the DNA polynucleotide comprises a sequencing adapter downstream of the T7 core promoter and a polyT-stretch downstream of the sequencing adapter and not overlapping with the sequencing adapter.

In another aspect, the present invention relates to a method for in vitro transcribing (IVT) RNA, said method comprising the steps of (a) providing a DNA polynucleotide according to the present invention as an IVT template and downstream thereof a nucleotide sequence encoding the RNA to be transcribed, and (b) in vitro transcribing said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.

In the context of the present invention, IVT refers to a process, wherein RNA is produced in vitro and thus, in a system that is neither a cell nor comprised in a cell. The term “cell” refers to a living organism or a unit of a multicellular organism or an explant thereof which can preferably be cultured. In vitro transcription requires an IVT template comprising a promoter and a template sequence downstream thereof, as well as ribonucleotide triphosphates and an RNA polymerase.

IVT can be used for linear amplification of a polynucleotide. Herein, the term “linear amplification” refers to increasing the amount of a polynucleotide over time in any relationship which resembles more a linear relationship than an exponential relationship at least for a certain period of time, for example 1 min, 10 min, 0.5 h, 1 h, 5 h, 10 h or 1 day. Linear amplification, as used herein, is advantageous over exponential amplification methods such as PCRfor certain applications, for example, but not limited to single-cell sequencing, in particular by reducing the amplification bias. Reducing an amplification bias is especially important when the starting material is scarce, such as the material obtained from a few cells or a single cell. Hence, in a preferred embodiment, the in vitro transcribed RNA is linearly amplified during IVT.

The term linear amplification of a polynucleotide, as used herein, comprises the amplification of the polynucleotide as RNA or DNA having the identical and/or complementary sequence of said polynucleotide. For in vitro transcribing (IVT) RNA, the DNA polynucleotide of the present invention is used as the IVT template. As regards the IVT template the same applies as it has been described above in connection with the DNA polynucleotide according to the present invention. Moreover, also the other features of such an IVT template can be as described above.

Hence, the DNA polynucleotide provided in step (a) comprises a T7 promoter sequence comprising a T7 core promoter sequence and a directly adjacent downstream flanking as defined above and a nucleotide sequence encoding the RNA to be transcribed, wherein said RNA is preferably an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA and/or CRISPR RNA.

In some embodiments, the nucleotide sequence encoding the RNA to be transcribed is an mRNA that encodes a transposase or a gene editing enzyme. Preferably, the transposase is a Sleeping Beauty transposase. Preferably, the genome editing enzyme is a CRISPR enzyme, for example, Cas9, Casl2b or Cpfl.

In some embodiments, the DNA polynucleotide which is used as an IVT template is particularly suitable for large-scale synthesis of RNA in vitro.

The IVT template is preferably linear, doubled-stranded and/or purified, even more preferably double-stranded, linearized and purified. In some embodiments, the DNA nucleotide of the invention is comprised in a plasmid such as a cloning and/or an expression vector. In this case, a linear IVT template can be obtained, for example, by linearizing a DNA plasmid comprising the IVT template by cutting the DNA plasmid using a restriction enzyme. A plasmid or vector, as used herein, refers to a double-stranded DNA molecule which can replicate within a bacterial host, preferably an E. coli strain. A plasmid or vector preferably comprises an origin of replication, a selection marker, such as a gene conferring resistance to an antibiotic, a cloning site such as restriction enzyme site, a recombination region, a promoter, an enhancer, a transcription termination site, a ribosomal binding site, a Shine Dalgarno and/or a Kozak sequence. Suitable plasmids as backbone are, for example, but not limited to pT7 FLAG (Merck), pT7 MAT (Merck), Gateway™ pDEST™ (Thermo Fisher) and pRSET (Thermo Fisher) of derivatives thereof. Furthermore, purification of the preferably linear and/or doubled- stranded IVT template is preferably performed using a spin-column such as a Monarch® PCR & DNA Cleanup Kit. In step (b), in vitro transcribing the IVT template is performed in the presence of ribonucleotide triphosphates which are preferably adenosine, uridine, cytidine and guanosine triphosphates or modified versions thereof. Furthermore, the T7 polymerase is preferably a T7 polymerase that is capable of binding to the T7 promoter.

In vitro transcribing the IVT template comprises using the IVT template as a template during transcription, i.e. the synthesis of an RNA molecule. In this case, said RNA has a nucleotide sequence that is identical to the sequence of the IVT template (identical to the coding strand and complementary to the template strand of the IVT template) except that instead of T nucleotides U nucleotides are comprised in the synthesized RNA molecule. Contrary, in case of reverse transcription an RNA is used as a template for the synthesis of a cDNA whereof the first strand has a sequence complementary to the sequence of the RNA and the second strand a sequence which is identical to the RNA except that instead of U nucleotides T nucleotides are comprised in the synthesized cDNA molecule.

In some embodiments, the DNA polynucleotide which is used as an IVT template is present in a high concentration in step (b), wherein the term “a high concentration” refers to a concentration of more than 30 ng/mΐ, preferably of more than 40 ng/pl.

As used herein, i.e. in the context of a polynucleotide, IVT template and/or target polynucleotide, the term “very low concentration” refers to a concentration of less than 1 ng/pl, preferably of less than 100 pg/pl, e.g. less than 50 pg/pl, and even more preferably of less than 10 pg/pl, in particular less than 1 pg/pl, or even less than 0.1 pg/pl, e.g. 0.05 pg/pl. Evidently, a very low concentration is more than 0, e.g. at least 0.005 pg/pl, 0.01 pg/pl or 0.05 pg/pl. Evidently, the term “pg” refers to “picogram”, the term “ng” to “nanogram”, and the terms “pi” or “ul” to “microliter”.

In some embodiments, the DNA polynucleotide which is used as an IVT template is present in a very low concentration in step (b), wherein the term “a very low concentration” refers to a concentration of less than 1 ng/pl, preferably of less than 100 pg/pl, e.g. less than 50 pg/pl and even more preferably of less than 10 pg/pl, in particular less than 1 pg/pl, or even less than 0.1 pg/pl, e.g. 0.05 pg/pl. Said DNA polynucleotide or IVT template may be a mix of different DNA molecules, e.g. cDNA molecules, wherein each of said DNA molecules comprises the T7 promoter of the invention, i.e. the same T7 core promoter and the same inventive upstream and downstream flanking regions. Said mix of DNA molecules may be obtained, for example, by reverse transcribing the mRNA from a few cells, i.e. less than 100 cells, preferably less than 10 cells, or, very preferably, from a single cell, and/or a liquid biopsy, with the reverse transcription primer comprising the inventive T7 promoter provided herein. The resulting cDNA (IVT template) of the single cells (or different samples) may be pooled if the reverse transcription primers have a barcode as described herein, but the concentration may still be very low as defined herein. The same principle applies to synthesizing the complementary strand of a target DNA polynucleotide, i.e. in a PCR reaction, from such few cells or a single cell and/or a liquid biopsy with a primer comprising the inventive T7 promoter and using the resulting library of DNA polynucleotides, i.e. double-stranded DNA molecules, which have incorporated said T7 promoter as IVT template, as described herein. Furthermore, the primers used for the PCR reaction may be complementary to different target DNA molecules, and thus the resulting IVT template may be a mix of different DNA molecules comprising the same inventive T7 promoter, which may be present at a very low concentration, as described herein.

Moreover, the IVT template comprising the T7 promoter of the invention may be obtained from at least one target polynucleotide that is present freely in a body fluid, such as blood, urine or cerebrospinal fluid. In case said target polynucleotide is a RNA, the IVT template may be obtained by reverse-transcription, and in case said target polynucleotide is a DNA, the IVT template may be obtained by synthesizing the complementary strand, i.e. in a PCR reaction, as described herein. If the target polynucleotide is a DNA, the IVT template may be also obtained by attaching and/or integrating the DNA polynucleotide of the invention harboring the inventive T7 promoter to the target polynucleotide via a transposon system i.e. Tn5 mediation transposition, as described herein. In particular, the IVT template may be present at a very low concentration as described herein.

As used herein, i.e. in the context of the use of the DNA polynucleotide of the invention as reverse transcription primer or (PCR) primer, i.e. for obtaining/generating an IVT template according to the invention and/or amplifying a target polynucleotide, e.g. a mRNA, a target polynucleotide or at least one target polynucleotide refers, in particular, to at least one polynucleotide to be analyzed in a sample, e.g. one or a few cells, a liquid biopsy, and/or a body fluid. In the context of reverse-transcription or synthesis of the complementary strand, the individual molecules of the target polynucleotide are not necessarily fully identical although they comprise the same or a very similar nucleotide sequence to which the reverse transcription primer or primer comprising the T7 promoter of the invention can bind. In particular, a very similar nucleotide sequence in this context may have at most 5, 4, 3, 2 or 1 nucleotide mismatches, e.g. due to SNPs, deletions and/or insertions, compared to the reference sequence which is complementary to a sequence comprised in the reverse-transcription primer or primer, and allows binding of the (reverse transcription) primer. For example, a target polynucleotide may refer to the mRNA molecules with a poly-A-tail in a sample, wherein the reverse transcription primer comprising a poly-T-stretch as provided herein binds to said poly-A-tail. However, a target polynucleotide may also refer, for example, to a polynucleotide comprising a unique nucleic acid sequence, and the reverse-transcription primer or primer comprising the complementary sequence of said unique sequence binds to said unique sequence. Furthermore, the target polynucleotide and the reverse-transcription primer or primer may be a mix of different polynucleotides, wherein the target polynucleotide is a plurality of polynucleotides which may comprise different unique nucleic acid sequences to which the reverse-transcription primer or primer mix binds.

In some embodiments, the target polynucleotide refers to a genomic DNA sequence to which the DNA polynucleotide of the present invention comprising the inventive T7 promoter is attached and/or integrated (i.e. covalently linked via the DNA backbone). In particular, said attachment and/or integration may be mediated by a transposase (transposon system), i.e a Tn5 transposase, e.g. as described in Harada (2019), Nat. Cell Biol. 21. In corresponding embodiments, the DNA polynucleotide of the invention further comprises a transposase binding sequence, i.e. a Tn5 transposase binding sequence which may be also known in the context of the Tn5 transpose as a “mosaic end”. Said transposase binding sequence is preferably downstream of the +1 to +8 downstream flanking region of the T7 promoter provided herein. In those embodiments, the individual molecules of the target polynucleotide may be structurally unrelated, wherein the target polynucleotide refers to genomic DNA sequences which are in proximity (e.g. within 5000 bp, 1000 bp, 500 bp, 200 bp or 100 bp, preferably within 200 bp) to a certain chromatin modification, e.g. a histone modification, and/or a DNA binding molecule, e.g. inter alia a transcription factor, an epigenetic modifier, CCCTC-binding factor (CTCF) or a polymerase. In particular, in the context of transposase-mediated attachment/integration of the T7 promoter of the invention, the DNA polynucleotide may be conjugated to a molecule (e.g. an antibody) which binds to a certain DNA binding molecule, as described herein. For example, the DNA polynucleotide of the invention may be conjugated to an antibody, e.g. via a linker such as a PEG linker, or to an antibody binding molecule such as protein A (which then binds to an antibody binding to a DNA-binding molecule) (Kaya-Okur (2019), Nat Commun. 10(1):1930). ChIL-Seq is a suitable, non-limiting, example for using the T7 promoter of the invention for transposon-mediated attachement/integration to genomic DNA sites (target polynucleotides) (Harada (2019), Nat. Cell Biol. 21). In ChIL-Seq, a T7 promoter sequence is fused at the 5’end to an Illumina adapter and a Tn5 recognition sequence and covalently coupled at the 3’end to an antibody with specificity for chromatin components i.e. specific histone modifications, and the Tn5 recognition sequence is loaded with Tn5 transposase. The cells (e.g. 100-1000 cells, or a single cell) containing the target polynucleotide of interest are then fixed, permeabilized and incubated with the Antibody-T7 promoter-Tn5 complex. Upon binding of the antibody to its target epitopes on the chromatin (DNA-binding molecules), the DNA polynucleotide harboring the T7 promoter sequence is covalently linked to the DNA backbone via Tn5 mediated transposition. The opposite DNA strand is repaired by incubation of the cells with DNA ligase, and DNA sequences adjacent to the integration sites (IVT template) are then in vitro transcribed into RNA by T7 polymerase. Conversion of the in vitro transcribed and amplified RNA into sequencing libraries followed by deep sequencing enables identification and/or quantification of the genomic antibody binding sites (i.e the target polynucleotide(s) in proximity to the DNA binding molecules that are recognized by the antibody).

Thus, a target polynucleotide or at least one target polynucleotide, as used herein, refers, in particular, to those polynucleotide molecules (e.g. in a sample) which are converted to an IVT template (comprising a T7 promoter according to the invention) as described herein (e.g. by reverse-transcription and/or synthesis of the complementary strand, or transposase-mediated integration/attachment). Therefore, the concentration, e.g. the very low concentration, of a target polynucleotide or at least one target polynucleotide, as used herein, refers, in particular, to the total concentration of those polynucleotide molecules (e.g. in a sample) which are converted to an IVT template.

In certain embodiments, i.e. in the context of reverse-transcription and/or amplification of RNA, the concentration of the IVT template is similar as the concentration of the corresponding target polynucleotide or at least one target polynucleotide, wherein “similar” may include a 5- fold, 3-fold, 2-fold, 1.5-fold, 1.2-fold, 1.1-fold or 1.05-fold difference, preferably a 2-fold or 1.5-fold difference. For example, a 2-fold difference is 10 pg/mΐ vs. 20 pg/mΐ, or 10 pg/mΐ vs. 5 pg/mΐ; and a 1.5-fold difference is 10 pg/mΐ vs. 15 pg/mΐ, or 10 pg/mΐ vs. 6.66 pg/mΐ.

Furthermore, when the inventive DNA polynucleotide is present in a very low concentration, i.e. in step (b) of the inventive IVT method provided herein, the DNA polynucleotide comprises preferably an upstream flanking region as described above in the context of the upstream region of a DNA polynucleotide according to the present invention.

In some embodiments, the in vitro transcribed RNA is a probe for hybridization with another RNA or DNA. Preferably, the probe is used for an in-situ hybridization method or southern blot. Preferably, the in-situ hybridization method is FISH. In case the in vitro transcribed RNA is to be used as a FISH probe, it is transcribed in the presence of fluorescently labeled ribonucleotides. A probe may be an antisense RNA which is complementary to the target oligo- or polynucleotide, for example an mRNA.

In some embodiments, the RNA is in vitro transcribed in the presence of a capping and/or polyA-tailing enzyme. In some embodiments, a 5’ cap is added at the 5’ end and/or a 3’ polyA tail is added at the 3’ end of the in vitro transcribed RNA.

A 5’ -cap, as used herein, preferably refers to a 7-methyl guanosine (m7G) cap structure at the 5^' end of an mRNA. The capping may be performed by enzyme-based capping following the transcription reaction (posttranscriptional capping) and/or by incorporation of a cap analog during transcription (co-transcriptional capping). Suitable cap analogues include 7-methyl guanosine (m7G) and 3^' O-me 7-meGpppG cap analog (ARC A). ARCA is methylated at the 3' position of the m7G, preventing RNA elongation by phosphodiester bond formation at this position. Thus, transcripts synthesized using ARCA contain 5^'-m7G cap structures in the correct orientation, with the 7-methylated G as the terminal residue. Suitable for posttranscriptional capping is, for example, the Vaccinia Capping System.

A poly-A-tail may be added during transcription and/or after transcription. Alternatively, a reverse PCR primer comprising a polyT-stretch can be used for amplifying the template sequence. The polyA tail can be also added after transcription using e.g. an E. coli polyA polymerase. The length of the added tail can be adjusted by titrating the polyA polymerase. As used herein, a polyA tail refers to a sequence comprising, preferably 5 to 300, covalently linked adenines.

In preferred embodiments, a 5’ cap is added at the 5’ end and a 3’ polyA tail is added at the 3’ end of the in vitro transcribed RNA. In some embodiments, the 5’ cap and/or the 3’ polyA tail is added while the RNA is in vitro transcribed. In some embodiments, the 5’ cap and/or the 3’ polyA tail is added after the RNA is in vitro transcribed. In some embodiments, the polyA tail is transcribed from an IVT template comprising a polyT- stretch at the 3 ’ end of the IVT template.

In some embodiments, the RNA is purified before and/or after a 5’ cap and/or a 3’ polyA tail is added.

In some embodiments, the nucleotide sequence encoding the RNA to be transcribed is an mRNA and the mRNA synthesized in vitro in step (b) is used to produce a recombinant protein in vitro or within a cell after providing an IVT mRNA. Preferably, the synthesis of the recombinant protein occurs in vitro.

In preferred embodiments, the nucleotide sequence encoding the RNA to be transcribed is an mRNA and the mRNA synthesized in vitro in step (b) is then transfected into a cultured cell. A cultured cell can be a mammalian cell line, an insect cell line, a yeast or a bacterium. Preferably, the cultured cell is a bacterium, even more preferably a cultured cell from E. coli. Transfection of a cell with an mRNA is particularly advantageous to only temporarily express the protein encoded by the mRNA in the cell and/or expressing said protein without changing the genome of the cell. Temporary expression is especially advantageous in case of a genome editing enzyme as the risk of unspecific genome-editing activity can be reduced.

Furthermore, the DNA polynucleotide according to the present invention can thus be used for producing, e.g. a recombinant, protein.

IVT is advantageous for amplifying a polynucleotide, wherein the amplified polynucleotide is DNA or RNA. Preferably, the polynucleotide to be amplified is RNA which is reverse transcribed into cDNA which in turn is amplified by IVT. Even more preferably, the RNA is an mRNA which is amplified as antisense RNA (aRNA).

In another aspect, the present invention relates to a method for transcribing RNA within a cultured cell, said method comprising the steps of (a) providing a DNA polynucleotide according to the present invention comprising a T7 promoter sequence and downstream thereof a nucleotide sequence encoding the RNA to be transcribed and (b) introducing the DNA polynucleotide into a cultured cell expressing a T7 polymerase.

In preferred embodiments, the RNA which is transcribed in a cultured cell is an mRNA which is further translated into a protein within the cultured cell. In preferred embodiments, the cultured cell wherein the RNA is transcribed is a bacterium.

In some aspect, the present invention relates to a cultured cell expressing a T7 polymerase comprising a DNA polynucleotide according to the present invention comprising a T7 promoter sequence and downstream thereof a nucleotide sequence encoding the RNA to be transcribed.

In preferred embodiments, the cultured cell comprising the DNA polynucleotide of the invention is a bacterium.

As regards the cultured cell and the DNA polynucleotide for transcription of an RNA within a cultured cell, the same applies as has been described above, in particular regarding the DNA polynucleotide of the invention, the cultured cell, the RNA to be transcribed and the production of a recombinant protein.

In particular, the DNA polynucleotide according to the present invention comprising an inventive T7 promoter provided herein may be used as a primer, i.e. for incorporating the sequence of the inventive DNA polynucleotide comprising the T7 promoter of the invention, into a copy of a target polynucleotide.

Thus, said DNA polynucleotide (primer) may be used for synthesizing the complementary strand of at least one target polynucleotide, wherein said synthesizing comprises annealing said DNA polynucleotide to said at least one target polynucleotide, in particular, thereby generating an IVT template according to the invention. In particular, said IVT template may be used in an inventive IVT method provided herein.

In preferred embodiments, the DNA polynucleotide according to the present invention is used as a reverse transcription primer. In particular, the DNA polynucleotide is used for reverse transcribing RNA to cDNA, thereby incorporating the sequence of the DNA polynucleotide at least partially into the cDNA.

As regards the use of the DNA polynucleotide of the invention as a primer or reverse transcription primer, the same applies as has been described above in connection with the T7 promoter, and in particular the downstream flanking region and/or the upstream flanking region of the T7 promoter, according to the invention.

Reverse transcription can be done using reverse transcriptases (RTs). RTs use an RNA as template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which may be used directly as a template for PCR. This combination, referred to as RT-PCR, allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA. Alternatively, the first strand cDNA can be made double-stranded using e.g. DNA Polymerase I and DNA Ligase. This is for example advantageous for cloning approaches without amplification. Suitable RT polymerases are, for example, Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV), reverse Transcriptase or, preferably a Superscript RT polymerase.

Moreover, the DNA polynucleotide according to the present invention may be used as a primer for synthesizing the complementary strand of a target DNA molecule, i.e. in a PCR reaction, thereby incorporating the sequence of the DNA polynucleotide of the invention at least partially into copies of the target polynucleotide. In particular, the synthesis of the complementary strand may be accompanied and/or followed by synthesizing the complementary strand of the strand having incorporated the DNA polynucleotide of the invention, thereby generating a double stranded DNA molecule comprising at least part of the target DNA molecule, and at one end the T7 promoter of the invention. This procedure may be considered as one cycle of a PCR reaction, wherein the DNA molecule of the invention is one PCR primer. Thus, typical PCR reaction conditions may be applied for this step, i.e. employing a DNA polymerase, for example, inter alia , a Taq polymerase. Preferably, said PCR reaction comprises only few cycles, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles, preferably 1, 2, 3, 4 or 5 cycles, preferably 1 or 2 cycles, preferably 1 cycle.

The target polynucleotide which is amplified by using a primer comprising the T7 promoter of the invention may be obtained, in particular, from a few cells (i.e. less than 100 cells), a single cell, a single cell equivalent, a liquid biopsy, and/or a body fluid, as described herein. In some embodiments, i.e. in the context of transposase mediate attachment/integration of the T7 promoter sequence, as described herein, the few cells may be 100 cells or more, for example 200, 400, 600, 800, 1000, 1500 or 2000 cells but less than 10000 cells. However, in general, the few cells, as described herein, are preferably less than 100 cells, more preferably less than 10 cells.

In certain embodiments, the target polynucleotide, i.e the target polynucleotide that is amplified, analyzed and/or sequenced, according to the invention is from a prokaryote or a eukaryote, preferably an animal, preferably a mammal, preferably a human. In particular, said target polynucleotide may be associated with a disease and/or is suspected to be associated with a disease, i.e. as described herein. In some embodiments, said target polynucleotide may be not from a virus, i.e. not from a virus that exists for less than 1000, 100 or 10 years. A liquid biopsy, as used herein, refers to the sampling of non-solid biological tissue, in particular, blood, and the analysis of at least one target polynucleotide comprised therein as described herein in the context of the invention. A liquid biopsy is mainly used as a diagnostic and monitoring tool, e.g. for diseases such as cancer, and may be largely non-invasive. In particular, a liquid biopsy may contain circulating tumor cells, e.g. inter alia , from metastatic breast, metastatic colon, and/or metastatic prostate cancer, and/or circulating tumor DNA. Furthermore, a liquid biopsy may contain circulating endothelial cells which are an indicator of vascular dysfunction or damage, e.g. associated with a heart attack. The presence or abundance of circulating tumor cells or circulating endothelial cells may be determined by a method, e.g. a sequencing method according to the invention, comprising amplifying target polynucleotides, e.g. mRNA molecules, from such single circulating cells by using a primer, e.g. a reverse transcription primer comprising an inventive T7 promoter, according to the invention.

Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream that is not associated with cells. In particular, because ctDNA may reflect the entire tumor genome, it may be useful in clinics, i.e. for diagnosing and/or monitoring cancer. The presence or abundance of circulating tumor DNA may be determined by a method, i.e. a sequencing method according to the invention, comprising amplifying target tumor DNA polynucleotides from a blood sample by using a primer comprising an inventive T7 promoter according to the invention.

Furthermore, a liquid biopsy may be from blood or amniotic fluid, and contain cell-free fetal DNA (cffDNA). cffDNA originates from placental trophoblasts and is fragmented when placental microparticles are shed into the maternal blood circulation. cffDNA fragments are approximately 200 base pairs (bp) in length which is significantly smaller than maternal DNA fragments. This difference in size allows cffDNA to be distinguished from maternal DNA fragments. Thus, the presence or abundance of circulating tumor DNA may be determined by a method, e.g. a sequencing method according to the invention, comprising amplifying target cell-free fetal DNA polynucleotides from a blood sample (or an amniotic fluid sample) by using a primer comprising an inventive T7 promoter according to the invention.

In particular, the ctDNA or cffDNA is linearly amplified by in vitro transcription employing the T7 promoter of the invention, and the IVT template is generated by few or one cycle(s) of PCR employing the inventive primer comprising said T7 promoter.

Thus, the DNA polynucleotide and/or the T7 promoter, i.e. the primer or reverse transcription primer of the invention may be used for diagnostic purposes, e.g. for diagnosing and/or monitoring cancer, a cardiovascular disease such as a heart attack, or a fetal gene defect or gene variant. As illustrated in the appended Examples, the sensitivity and accuracy of single cell sequencing methods can be increased by employing a primer comprising an inventive T7 promoter provided herein for capturing target polynucleotides and amplifying them by in vitro transcription. This increase in sensitivity and accuracy may be particularly useful for diagnostic approaches, e.g. to determine certain cell states, e.g. of circulating tumor cells or leukemic cells, more reliably. In fact, as illustrated in the appended Examples, cell state determinants such as transcription factors and chromatin binders, which are missed when using a conventional T7 promoter, are detected by the improved sequencing method of the invention employing a reverse transcription primer comprising an inventive T7 promoter provided herein.

Thus, in a further preferred aspect, the invention relates to a method for amplifying a target polynucleotide, wherein said method comprises the steps of

(a) synthesizing the complementary strand of a target polynucleotide, wherein said synthesizing comprises annealing a DNA polynucleotide comprising the T7 promoter sequence according to the invention to said target polynucleotide, thereby obtaining an IVT template comprising said T7 promoter and a nucleotide sequence downstream thereof, i.e. wherein said nucleotide sequence reflects the target polynucleotide sequence, and

(b) in vitro transcribing said IVT template according to the IVT method of the invention, thereby amplifying said target polynucleotide. In particular, said amplification is a linear amplification as described herein. In particular, reflecting the target polynucleotide sequence refers to the target polynucleotide sequence and the complementary sequence or antisense sequence thereof, and/or a well attributable fragment thereof. A fragment can be attributed to the target polynucleotide sequence by methods well known in the art, as described herein or illustrated in the appended Examples, e.g. by a sequence alignment algorithm.

In certain embodiments, the target polynucleotide is an RNA which is reverse-transcribed into cDNA. In particular, said RNA is thereby amplified as aRNA.

In preferred embodiments, the IVT template is obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from less than 100 cells, preferably less than 10 cells, preferably a single cell.

In the context of the present invention, the cell containing an RNA that is to be reverse transcribed to cDNA or a target polynucleotide that is to be amplified, and/or a polynucleotide that is to be analyzed and/or sequenced may be a prokaryotic cell or an eukaryotic cell, preferably an eukaryotic cell, preferably an animal cell, preferably a mammalian cell, preferably a human cell. In particular, the human and/or animal cell may be associated with a disease and/or be suspected to be associated with a disease, e.g. inter alia cancer or a cardiovascular disease. Said cancer may be, in particular, characterized by metastatic cells, circulating tumor cells and/or blood cancer cells / leukemic cells.

Furthermore, the IVT templated may be obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide, wherein said RNA and/or at least one target polynucleotide is present at a concentration of less than 1 ng/pl, preferably of less than 100 pg/mΐ, e.g. less than 50 pg/mΐ, and even more preferably of less than 10 pg/mΐ, in particular less than 1 pg/mΐ, or even less than 0.1 pg/mΐ, e.g. 0.05 pg/mΐ. Moreover, the IVT template may be obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from a liquid biopsy and/or a body fluid.

In preferred embodiments, at least one target polynucleotide is amplified from less than 100 cells, preferably less than 10 cells, preferably a single cell, wherein the T7 promoter further comprises the sequence AATT directly upstream of the T7 core promoter sequence. Preferably, the T7 promoter further comprises a poly-T-stretch as described herein. In particular, mRNA molecules from less than 100 cells, preferably less than 10 cells, preferably a single cell corresponding to at least 5000, preferably at least 7000, preferably at least 9000 different genes may be amplified. Preferably, mRNA molecules from a single cell corresponding to at least 9000 different genes may be amplified. An mRNA molecule corresponding to a gene refers, in particular, to a transcript of said gene. Evidently, the maximum amount of different genes detected and/or amplified is limited by the genes expressed in a cell, and may thus vary between individual cells or samples.

Herein, a gene may refer to the genomic sequence of a gene comprising the exons of said gene. Moreover, said genomic sequence may comprise the intronic regions and/or, in some cases, the regulatory regions of said gene such as a promoter. Moreover, a gene may refer to a cDNA or a coding sequence. In particular, in the context of RNA sequencing, transcripts, reverse transcription, mRNA and/or cDNAs, a gene may preferably refer to the part of the gene that is transcribed (i.e. a cDNA).

In preferred embodiments, the RNA which is reverse transcribed into cDNA is obtained from a single cell or a single cell equivalent. A single cell equivalent, as used herein, is a fraction of a sample comprising RNA, wherein the concentration of the RNA and/or the total amount of RNA is similar to what is obtained from a single cell by methods used in the art. The amount of RNA obtained from a single cell is about 10 to 30 pg and the amount of mRNA obtained from a single cell is about 0.1 to 1.5 pg. Moreover, a single cell equivalent may be a sample of a liquid biopsy and/or a body fluid, such as blood, urine or cerebrospinal fluid, comprising a similar amount and/or concentration of RNA, mRNA and/or DNA as a single cell. In some embodiments, a single cell equivalent may comprise about 0.1 to 1.5 pg and/or at least one target polynucleotide, e.g. at least one target mRNA and/or at least one target DNA, at a very low concentration as described herein. In some cases, a sample of a body fluid and/or liquid biopsy may be concentrated or diluted and/or subsampled to comprise about 0.1 to 1.5 pg and/or at least one target polynucleotide at a very low concentration, as described herein.

A polyT-stretch is particularly useful when the DNA polynucleotide of the invention is used as a reverse transcription primer for reverse transcribing mRNA to cDNA. Hence, in a preferred embodiment, the DNA polynucleotide comprising the T7 promoter of the invention and further comprising a polyT-stretch is used as a primer for reverse transcribing mRNA to cDNA, wherein the polyT-stretch binds to the polyA tail of the mRNA. Similarly, a polyT-stretch is useful for reverse-transcribing a non-coding RNA comprising a polyA tail to cDNA.

In a preferred embodiment, a cDNA comprising the T7 promoter of the invention and downstream thereof the antisense sequence of an mRNA is used as IVT template for in vitro transcribing antisense RNA (aRNA). Preferably, the aRNA is thereby linearly amplified.

In certain embodiments, the DNA polynucleotide comprising the T7 promoter of the invention is used for linearly amplifying RNA from cDNA by in vitro transcription.

In a preferred embodiment, the DNA polynucleotide comprising the T7 promoter comprising the downstream flanking region of the invention and the upstream flanking region of the invention is used for linearly amplifying RNA from cDNA by in vitro transcription.

In a particularly preferred embodiment, cDNA is generated from RNA by using the DNA polynucleotide comprising the T7 promoter comprising the downstream flanking region of the invention and the upstream flanking region of the invention as reverse transcription primer. Preferably, the RNA is obtained from less than 100 cells, preferably less than 10 cells, very preferably from a single cell.

The T7 promoter comprising the downstream flanking region of the invention and the upstream flanking region of the invention is particularly suitable for in vitro transcribing RNA, when the IVT template concentration is low, i.e. very low as described herein. The IVT template concentration is low, for example, when the IVT template is a cDNA which is reverse transcribed from an RNA obtained from a single cell. Hence, the DNA polynucleotide of the invention comprising a T7 promoter comprising a downstream flanking region of the invention and an upstream flanking region of the invention is particularly useful for in vitro transcribing RNA from cDNA, wherein the cDNA has been reverse transcribed from RNA present in a very low concentration and/or derived or obtained from less than 100 cells, preferably a single cell. Moreover, said DNA polynucleotide comprising a T7 promoter comprising a downstream flanking region of the invention and an upstream flanking region of the invention is very useful for in vitro transcribing RNA from a DNA polynucleotide, i.e. a double stranded DNA molecule, wherein said DNA polynucleotide has been generated from a target DNA polynucleotide that is present in a very low concentration and/or derived or obtained from less than 100 cells, preferably a single cell.

Evidently, a polynucleotide that is derived or obtained from a cell, has been contained in said cell, and/or is contained in such a cell.

In a particularly preferred embodiment, aRNA is linearly amplified by in vitro transcribing aRNA from a cDNA IVT template, wherein the cDNA is obtained by reverse transcription from an mRNA, wherein the DNA polynucleotide of the invention is comprised in the reverse transcription primer, and wherein the mRNA is obtained from a single cell.

In a preferred embodiment, the method for in vitro transcribing RNA further comprising the steps of reverse transcribing an RNA to cDNA and subsequently transcribing the cDNA to RNA, wherein the DNA polynucleotide of the invention is used as reverse transcription primer and the cDNA is the IVT template. Preferably, the reverse transcribed RNA is an mRNA, the DNA polynucleotide comprises a poly-T-stretch, preferably downstream of the T7 core promoter, and the in vitro transcribed RNA is an antisense RNA (aRNA).

As regards the initial reverse transcription step in certain embodiments of the method of the invention for in vitro transcribing RNA, the same applies as it has been described above in connection with use of the DNA polynucleotide as reverse transcription primer.

In preferred embodiments, a second strand of the cDNA comprising the T7 promoter of the invention is synthesized upon first strand cDNA synthesis.

In certain embodiments, the linearly amplified RNA is further reverse transcribed to cDNA, preferably by using random primers for first strand synthesis and the DNA polynucleotide of the invention comprising a polyT-stretch as primer for second strand synthesis. The cDNA derived from linearly amplified RNA can again be used as an IVT template to again in vitro transcribe RNA.

In some embodiments, the cDNA comprising the T7 promoter of the invention and downstream thereof a nucleotide sequence encoding preferably an RNA is used as IVT template for in vitro transcribing RNA. Preferably, the RNA is thereby linearly amplified. Hence, the DNA polynucleotide of the invention can be used for, preferably linearly, amplifying a nucleotide sequence which is complementary to a nucleotide sequence comprised in said DNA polynucleotide.

Furthermore, in the context of the present invention, the presence and/or abundance of a target polynucleotide in sample such as a liquid biopsy, a sample from a body fluid, a few cells and/or a single cells, as described herein, may be determined by employing the inventive IVT and T7 promoter based amplification methods provided herein. Moreover, the amount of the amplified target polynucleotide(s) (e.g. aRNA) may be determined by methods known in the art and/or as described herein, e.g. by quantitative PCR, digital PCR and/or next-generation sequencing. Further methods to detect the amplified target polynucleotide(s) may be also used, e.g. fluorescence in-situ hybridization (FISH) and/or dCas9-based imaging.

In a further aspect, the invention relates to a method for amplifying a target polynucleotide, wherein said target polynucleotide is a genomic DNA sequence, and wherein said method comprises the steps of

(a) attaching and/or integrating a DNA polynucleotide comprising the T7 promoter sequence according to the invention to said target polynucleotide by transposition, i.e. Tn5 transposition, thereby obtaining an IVT template comprising said T7 promoter and a nucleotide sequence downstream thereof, i.e. wherein said nucleotide sequence reflects the target polynucleotide sequence, and

(b) in vitro transcribing said IVT template according to the IVT method of the invention, thereby amplifying said target polynucleotide.

As regards the in vitro transcription (step b), the amplification of the target polynucleotide, and “reflecting the target polynucleotide sequence”, the same applies as is described herein in the context of further methods for amplifying a target polynucleotide.

In particular, employing a primer or reverse transcription primer comprising a T7 promoter with an inventive and defined downstream flanking region, as provided herein, allows to compare the abundances of target polynucleotides (e.g. non-poly-A RNA or DNA) with improved sensitivity and/or accuracy. The same applies for T7-promoter based transposase-mediated amplification of genomic DNA target polynucleotides (e.g. which are in proximity to a certain epigenetic modification or DNA-binding molecule).

In particular, the improved accuracy is due to the improved efficiency of amplifying the target polynucleotide, and furthermore, due to the lack of an amplification bias. Such an amplification bias may occur when only a core T7 promoter or a T7 core promoter with less than 8 defined directly adjacent downstream bases is used because in such a case the efficiency of the T7 promoter may vary between different target polynucleotides since they may constitute a (random) part of the downstream flanking region. The T7 promoter of the invention comprising an optimized and defined downstream flanking region is thus particularly useful for detecting and/or quantifying target polynucleotides in a sample, i.e. from less than 100 cells, preferably less than 10 cells, preferably a single cell, a single cell equivalent, a body fluid, and/or a liquid biopsy as described herein. In particular, for quantification of a plurality of target polynucleotides, e.g. a single cell transcriptome, an inventive sequencing method employing an inventive primer provided herein may be used.

In particularly preferred embodiments, the IVT method using a DNA polynucleotide according to the present invention is part of a sequencing method.

Herein, the term "sequencing" refers to approaches aiming at determining the identity of at least one nucleotide, preferably most nucleotides, even more preferably all nucleotides in a given nucleotide sequence. Furthermore, the term “sequencing method” refers to a method for determining the partial or full sequence(s) of a polynucleotide and/or to determine the relative abundance of identical molecules of said polynucleotide.

Examples for sequencing methods and/or commercial suppliers of sequencing methods include Sanger sequencing, pyrosequencing, SOLiD sequencing, Illumina sequencing, Ion Torrent sequencing, single molecule real time sequencing and Nanopore sequencing. A sequencing method may be a first, next (second) or third generation sequencing method. Preferred are next or third generation sequencing methods. A preferred next-generation sequencing technique is Illumina sequencing. Preferably, the sequencing method is suitable for single cell sequencing. Suitable single cell sequencing techniques are for example based on SMART-seq2, CEL-seq2, inDrop, MARS-seq, Drop-seq, STRT, Quartz-seq, LIANTI, CHIL-Seq, Dam-seq (e.g. scDam&T-seq) and the sci-L3 method. Particularly suitable are sequencing methods that comprise an in vitro transcription step such as CEL-seq, CEL-seq2, inDrop, MARS-seq, LIANTI, CHIL-Seq, Dam-seq (e.g. scDam&T-seq), and sci-L3. A preferred third generation sequencing method is Nanopore sequencing which allows direct sequencing of RNA and/or DNA in the absence of a library preparation step comprising PCR based amplification of reverse-transcribed aRNA.

Hence, in some embodiments, the DNA polynucleotide of the present invention is used to reverse transcribe a target polynucleotide, e.g. an RNA, amplify it, i.e. by IVT, and then subject the amplified RNA directly and/or after reverse transcription to sequencing.

As regards the DNA polynucleotide the same applies as described above. Thus, in particularly preferred embodiments, the DNA polynucleotide used for the sequencing method comprises a polyT-stretch to capture mRNAs which are then amplified as antisense RNAs (aRNAs). Thus, the entire sequence of RNAs, including the sequence of the respective poly(A) tails, can be determined by sequencing the synthesized cDNA library. In further particularly preferred embodiments, the DNA polynucleotide of the invention used for the sequencing method further comprises a barcode and/or a unique molecular identifier (UMI) and preferably a polyT-stretch.

In preferred embodiments, the sequencing method is suitable for scarce material, in particular for less than 10.000 cells, preferably less than 100 cells, preferably less than 10 cells. In a very preferred embodiment, the sequencing method is a single cell sequencing method.

In preferred embodiments, the invention relates to a method for determining the partial or full sequence(s) of a polynucleotide, i.e. a target polynucleotide, and/or the relative abundance of identical molecules of said polynucleotide comprising the steps of (a) synthesizing a DNA strand which is complementary to the polynucleotide that is to be sequenced (target polynucleotide) or the complementary strand thereof comprising annealing the DNA polynucleotide of the invention (to said target polynucleotide), and (b) transcribing the polynucleotide that is to be sequenced (target polynucleotide) or the complementary strand thereof into RNA by using a T7 polymerase.

In preferred embodiments, the present invention relates to a method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene comprising the steps of (a) reverse transcribing an RNA into a first strand of a cDNA comprising annealing the DNA polynucleotide according to the present invention; and (b) transcribing the cDNA into aRNA by using a T7 polymerase.

As regards the reverse transcription step (step (a)) of the method, the same applies as it has been described above in connection with reverse transcribing RNA to cDNA, synthesizing the complementary strand of a target polynucleotide and/or the use of the DNA polynucleotide of the invention as a primer or reverse transcription primer. Furthermore, the term “annealing the DNA polynucleotide” refers herein to the use of the DNA polynucleotide of the invention as a primer for reverse transcribing or synthesizing the complementary strand of the polynucleotide the sequence of which is to be determined (target polynucleotide).

In a preferred embodiment, the method further comprises a step (a’) of synthesizing a second strand of the first strand of the cDNA obtained from step (a) or during the method for amplifying a target polynucleotide according to the invention.

Thus, in a further preferred aspect, the invention relates to a method for determining the partial or full nucleotide sequence(s) and/or abundance of at least one target polynucleotide comprising the steps of

(a) amplifying at least one target polynucleotide according to the method for amplifying a target polynucleotide according to the invention, and

(b) determining the partial or full nucleotide sequence(s) and/or abundance of said at least one amplified target polynucleotide as described herein or illustrated in the appended Examples.

In particular, determining the abundance of at least one target polynucleotide may comprise counting the nucleotide sequences corresponding to said target polynucleotide. Methods for determining and normalizing the counts are well known in the art, and described herein and in the appended Examples. Moreover, the abundance of at least one mRNA may further refer to the transcript level of the corresponding gene.

Thus, the invention also relates to a method for determining the transcript level of at least one gene comprising the steps of

(a) amplifying at least one target polynucleotide according to the method for amplifying a target polynucleotide according to the invention, wherein said target polynucleotide is an mRNA corresponding to said at least one gene, and

In preferred embodiments, the partial or full nucleotide sequence(s), the abundance of at least one target polynucleotide and/or the transcript level of at least one gene is determined in a sample comprising less than 100 cells, preferably less than 10 cells, preferably a single cell. Moreover, the mRNA and/or at least one target polynucleotide may be present at a concentration of less than 1 ng/mΐ, preferably of less than 100 pg/mΐ, e.g. less than 50 pg/mΐ, and even more preferably of less than 10 pg/mΐ, in particular less than 1 pg/mΐ, or even less than 0.1 pg/mΐ, e.g. 0.05 pg/mΐ.

As illustrated in the appended Examples, the expression of at least about 9750 genes can be detected in a single cell (e.g. a K562 cell) with an exemplary sequencing method according to the invention employing an inventive reverse transcription primer provided herein (e.g. SEQ ID NO: 15) comprising an T7 promoter of the invention (e.g. SEQ ID NO:20).

Thus, the partial or full nucleotide sequence(s) and/or the abundance of at least 5000, preferably at least 7000, preferably at least 9000 different target polynucleotides and/or the transcript level of at least 5000, preferably at least 7000, preferably at least 9000 genes may be determined according to the sequencing method of the invention. In particular, at least 5000, preferably at least 7000, preferably at least 9000 transcripts of unique genes may be detected in a single cell. Moreover, at least 9100, 9300 or 9500 transcripts of unique genes may be detected in a single cell.

In preferred embodiments, the sequencing method of the invention, e.g. the method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene comprises a step (c) of generating a double-stranded cDNA from the aRNA of step (b), and/or a step of sequencing the aRNA of step (b) and/or the cDNA of step (c).

In some embodiments, the sequencing method of the invention further comprises a step (c’) of synthesizing the second strand of the cDNA of step (c) upon synthesis of the first strand of the cDNA.

In preferred embodiments, the method further comprises a step (d) of amplifying the cDNA of step (c) by PCR, wherein a library preparation primer binds to the sequencing adapter. Preferably, the sequencing method further comprises a step of sequencing the amplified cDNA of step (d).

In preferred embodiments, the partial or full nucleotide sequence(s) of an mRNA and/or the transcript level of at least one gene comprising a coding sequence is determined, wherein the DNA polynucleotide of the invention comprises a polyT-stretch and a sequencing adapter, wherein the polyT-stretch binds to the poly-A-tail of the mRNA.

In a preferred embodiment, the polynucleotide that is to be sequenced (target polynucleotide) is derived or obtained from less than 10000 cells, preferably, less than 100 cells, preferably less than 10 cells. In a further preferred embodiment, the polynucleotide to be sequenced (target polynucleotide) is derived or obtained from a single cell and/or a single cell equivalent. Preferably, the polynucleotide that is to be sequenced (target polynucleotide) is derived from and/or obtained from a single cell. Furthermore, the polynucleotide to be sequenced may be derived or obtained from a body fluid, i.e. a single cell equivalent obtained from a body fluid, as described herein.

In a very preferred embodiment, the sequencing method of the invention further comprises a step of analyzing the sequencing data, wherein the analysis comprises a sequence alignment, counting reads, and/or normalizing read counts. In a further aspect, the present invention relates to the use of the DNA polynucleotide according to the present invention in a sequencing method, e.g. for determining the partial or full nucleotide sequence(s) of an RNA contained in a single cell or single cell equivalent and/or the transcript level of at least one gene of a single cell or single cell equivalent.

As regards the DNA polynucleotide and the determination of the partial or full nucleotide sequence(s) of an RNA contained in a single cell or single cell equivalent and/or the transcript level of at least one gene of a single cell or single cell equivalent, the same applies as described above.

The present invention further relates to a kit comprising the DNA polynucleotide of the invention, one or more modified and/or unmodified ribonucleotide triphosphate(s), library preparation primer(s), sequencing primer(s), and/or a microfluidic chip.

The kit can further comprise an enzyme for 5’ capping of RNA and/or an enzyme for poly-A- tailing of RNA, and/or a manual describing an in vitro transcription method and/or a sequencing method comprising an IVT step using said kit. As regards the DNA polynucleotide and modified and/or unmodified ribonucleotide triphosphate(s) of the kit, the same applies as it has been described above in connection with the DNA polynucleotide according to the invention.

As regards the use of the kit, the same applies as it has been described above in connection with the use of the DNA polynucleotide according to the invention. In some embodiments, the polynucleotide of the invention is an RNA polynucleotide. As regards the RNA polynucleotide of the invention, the same applies as it has been described in the context of a DNA polynucleotide.

In a preferred embodiment, the RNA polynucleotide of the invention is reverse-transcribed to DNA, in particular before its use as a reverse transcription primer, for IVT and/or a sequencing method according to the invention.

Brief description of the figures

Figure 1. Scheme of 5’RACE-Seq for investigating the efficiency of different T7 promoter sequences. A double stranded DNA (dsDNA) library (SEQ ID NO:2), wherein each DNA polynucleotide comprised a T7 core promoter sequence, followed by a guanine at position +1 and a randomized nucleotide sequence at positions +2 to +16, was transcribed in vitro using a T7 RNA polymerase. The resulting 210 nucleotides long RNAs were reverse transcribed, and the 5’ end of the respective cDNA converted into a library for deep sequencing. The activity of the T7 promoter sequences was determined by counting the reads comprising a respective flanking region directly adjacent downstream the T7 core promoter sequence. The DNA IVT template was amplified by PCR and sequenced for normalization of the read counts.

Figure 2. Relative abundances of T7 promoter sequence variants from the TSS to position +16 directly adjacent downstream the T7 core promoter sequence (each with a G at position +1) and grouped by the respective dinucleotide sequence at positions +2 and +3. Promoters with three guanines at positions +1 to +3 (GGG) showed the highest activity on average as detected by 5’RACE-Seq.

Figure 3. Normalized average nucleotide compositions of T7 promoter sequence variants from positions +2 to +16 in amplified (i.e. in vitro transcribed) RNAs, determined by 5’RACE-Seq. The overlapping part of the extended initiation bubble, which extends from positions -4 to +7, is highlighted in light grey.

Figure 4. Activity of T7 promoter variants carrying nucleotide sequences from positions +4 to +8 determined by 5’RACE-Seq shown as log2 fold change (FC) (relative abundances of individual sequence motifs). All promoters contained a G at positions +1 to +3. A high correlation (R²) of 0.98 was observed between two independent experiments denoted as RepA and RepB. Highlighted are exemplary T7 promoter sequences with high, mid, or low activity. Figure 5. Linear amplification of RNA by in vitro transcription. In vitro transcription was performed to compare the activity of T7 promoter variants with a G at positions +1 to +3 followed by different nucleotide sequences at positions +4 to +8 with either a high, intermediate or low rank as determined by 5’RACE-Seq as shown in Table 1. A 410 nucleotides long RNA was in vitro transcribed for the indicated time points using the respective promoter variant (SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8). Shown is the resulting fold amplification of template DNA. Error bars represent standard deviation for triplicate experiments.

Figure 6. Comparison of the IVT activity of T7 promoter variants with different 5’RACE-Seq ranks as shown in Table 1. All T7 promoter variants comprised a G at positions +1 to +3 and different nucleotide sequences at positions +4 to +8 and were used to in vitro transcribe a 410 nucleotides long RNA for 2h. Shown is the fold amplification relative to the template DNA. Indicated below is the +4 to +8 RACE-Seq rank as shown in Table 1. Results are shown from left to right for SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:3, and SEQ ID NO:4. Error bars represent standard deviation for triplicate experiments

Figure 7. Comparison of the IVT activity of two T7 promoter variants using different IVT DNA template concentrations (SEQ ID NO:4 and SEQ ID NO: 8). IVT was performed for 2h.

Figure 8. Comparison of the IVT activity of a T7 promoter variant with a 5’RACE-Seq rank as shown in Table 1 with or without an AT -rich 4 nucleotides long upstream flanking region. IVTs were performed for 2h using T7 promoter variants of the indicated 5'RACE-Seq rank. Shown is the resulting fold amplification of template DNA for SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 8 (from left to right). The upstream flanking region was GAATT located directly adjacent upstream the T7 core promoter (comprised in SEQ ID NO:5).

Figure 9. An AT -rich upstream flanking region boosts the IVT activity of the T7 promoter in an IVT template that mimics the structure of CEL-seq2 derived cDNA, in particular at very low IVT template concentrations. In the left panel, a 410 nucleotides long RNA was in vitro transcribed for 2h from 1 nanogram of standard IVT templates (SEQ ID NO:4 and SEQ ID NO:5). In the middle panel, the RNA was in vitro transcribed for 2h from 1 nanogram (50 pg/pl) of 305 nucleotides long CEL-seq cDNA mimics (SEQ ID NO: 10 and SEQ ID NO: 11). In the right panel, the RNA was in vitro transcribed for 15h from 1 picogram (0.05 pg/mΐ) of those CEL-seq cDNA mimics. The T7 promoter in all IVT templates further comprised a directly adjacent downstream flanking region with the sequence “GGGATAAT”. Figure 10. A T7 promoter of the present invention, in particular with a respective upstream flanking region improves IVT from a dsDNA that mimics the structure of CEL-seq2 derived cDNA. Exemplarily, the relative activity of the T7 promoter as found in the CEL-seq2 adaptor (see SEQ ID NO:9) is compared to the T7 promoter with rank #4 (see SEQ ID NO: 10) in the 5’RACE-Seq data as shown in Table 1, and the latter in combination with the directly adjacent upstream flanking region with the sequence GAATT (see SEQ ID NO: 11). The template was pre-synthesized as double stranded DNA to monitor the specific effect of the promoter sequence, independent of the mRNA capturing step during CEL-seq2. 1 picogram template was in vitro transcribed for 15h. The data for “CEL-seq + #4” and “CEL-seq up + #4” are the same as shown in the right panel of Figure 9.

Figure 11. T7 promoter in combination with specific directly adjacent upstream flanking regions improves aRNA yield during CEL-seq2 from 10 single cells. CEL-seq2 experiments were performed until RNA amplification using the reverse transcription primers with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 14 or SEQ ID NO: 16 (from left to right). The 5’RACE-Seq rank of the T7 promoter sequence is indicated as shown in Table 1. “upl” refers to the upstream flanking region GAATT (comprised in SEQ ID NO: 13 and SEQ ID NO: 15) and “up4” refers to the upstream flanking region AATTG (comprised in SEQ ID NO: 14 and SEQ ID NO: 16). Due to some sequence similarity of the directly adjacent downstream flanking region of the T7 core promoter comprised in the original CEL-seq2 primer and the T7 promoter with rank #66, only two nucleotides (AG) were inserted after position +3 in case of the T7 promoter sequence with rank #66 (see SEQ ID NO: 15), in contrast to the variant with rank #4 (see SEQ ID NO: 13), wherein six nucleotides were inserted (ATAATG). The term “CEL-seq” indicates the original CEL-seq2 reverse transcription primer (SEQ ID NO: 12). Error bars represent standard deviation for triplicate experiments.

Figure 12. The combination of a directly adjacent up- and downstream region of the T7 improves IVT in a single cell RNA sequencing experiment based on CEL-seq2. CEL-seq2 was performed from single K562 cells using the indicated primers (SEQ ID NO: 12 and SEQ ID NO: 15). After reverse transcription and second strand synthesis cDNA from 10 cells was pooled and in vitro transcribed for 15h. Purified RNA was fragmented and quantified on a Tapestation (Agilent). Error bars represent the standard deviation from triplicate experiments.

Figure 13. CEL-Seq2 workflow. Shown on top is a schematic of the CEL-Seq2 reverse transcription primer (T7 = T7 promoter; Illumina A = partial sequencing adapter A; UMI = unique molecular identifier; BC = cellular barcode). Polyadenylated RNA from single cells is captured via the oligo d(T) terminus of the CEL-Seq2 reverse transcription primer. After reverse transcription (RT) and second strand synthesis, mRNA sequences are amplified by in vitro transcription (aRNA production). Antisense RNA (aRNA) is fragmented, and reverse transcribed using a random hexamer primer fused to partial sequencing adapter B (Illumina B). Sequencing adapters are completed, and the library is amplified in a final PCR step.

Figure 14. A modified CEL-Seq2 reverse transcription primer (SEQ ID NO: 15; CEL-Seq+) harboring a modified T7 promoter (SEQ ID NO:20) significantly improved CEL-Seq2-based RNA-Sequencing of single cells, i.e. by enhancing the efficacy of the linear amplification of cDNA. Specifically, a significantly increased number of genes was detected with CEL-Seq+. With the modified CEL-Seq2 reverse transcription primer (SEQ ID NO: 15) 9749 genes were detected per cell on average (n=24 cells), whereas only 8281 genes were detected per cell on average (n=14 cells) with the conventional CEL-seq2 reverse transcription primer (SEQ ID NO: 12) (left plot). Moreover, CEL-SEQ+ significantly increased the number of detected molecules. With the modified CEL-Seq2 reverse transcription primer (SEQ ID NO: 15), 85066 unique molecular identifiers (UMIs)/transcripts per cell were detected on average, whereas only 53541 UMIs per cell were detected on average with the conventional CEL-seq2 reverse transcription primer (SEQ ID NO: 12) (right plot). Statistical analysis was performed using the Mann-Whitney Wilcoxon test.

Figure 15. Average UMI count per gene (left plot) and corresponding coefficient of variation (CV) for UMI counts (right plot) in CEL-Seq2 employing the conventional CEL-seq2 reverse transcription primer (SEQ ID NO: 12) and CEL-Seq+ employing the modified CEL-Seq2 reverse transcription primer (SEQ ID NO: 15) . Shown are all genes with more than 1 average UMI per cell in both assays (n=7904). Genes are independently sorted by expression rank. 14 cells from CEL-Seq+ were randomly chosen for comparison with 14 cells from CEL-Seq2. Error bars show the standard deviation between cells. Thick lines refer to the average UMI counts (left) or CV (right) of all 14 cells interpolated across all genes assayed with either CEL- Seq2 or CEL-seq+ (as marked by the arrows).

Figure 16. Genes from deep sequenced bulk K562 RNA-Seq (Prost (2015), Nature 525) were sorted into quartiles by expression level with the lowest expressed genes in the first quartile and the highest expressed genes in the fourth quartile. Shown are the numbers of genes from the indicated bulk quartiles that were detected in individual cells by CEL-Seq2 (with the reverse transcription primer shown in SEQ ID NO: 12) or CEL-Seq+ (with the reverse transcription primer shown in SEQ ID NO: 15). Within each of the four quartile plots, CEL-Seq2 is shown on the left, and CEL-Seq+ on the right.

Figure 17. Shown are the numbers of genes of different categories detected with CEL-Seq2 (with the reverse transcription primer shown in SEQ ID NO: 12) or CEL-Seq+ (with the reverse transcription primer shown in SEQ ID NO: 15) in individual cells. The first category encompasses genes that are differentially expressed throughout chronic myeloid leukemia (CML) disease progression (Prost (2015), Nature 525; left plot), the second category encompasses genes with the GO association “DNA binding transcription factor (TF)” (middle plot), and the third category encompasses genes with the GO association “chromatin binding” (right plot). Within each of the three gene category plots, CEL-Seq2 is shown on the left, and CEL-Seq+ on the right. Whiskers reach to 1.5x IQR away from the lst/3rd quartile.

Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.

Examples

Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

Example 1. Assessment of the strength of T7 core promoter sequences with different directly adjacent flanking regions using 5’RACE-Seq

5 ’RACE- Sea

A saturated randomized 5' RACE-Seq screen was performed to determine the self-transcription activity of T7 promoter sequences comprising the T7 core promoter, a guanine at position +1 (TSS) and different combinations of nucleotides at positions +2 to +16. In particular, the 5’ end sequence of over 100 million 210 nucleotides long RNAs transcribed from said randomized promoter sequences comprised in a 500 nucleotides long dsDNA polynucleotide library (lOng; scale of 10¹⁰ (10 billion) molecules of the +2 to +16 randomized T7 promoter template) was determined to interrogate the influence of the directly adjacent downstream flanking region of the core promoter on the transcriptional activity of the T7 promoter. A scheme of the applied experimental procedure of 5' RACE-Seq is shown in Figure 1. 5' “RACE-Seq” refers to a rapid amplification of cDNA ends and subsequent sequencing using a next-generation-sequencing method (deep sequencing). A “saturated” screen refers to a screen of virtually any possible combination of nucleotides at positions +2 to +16 contained in a 5’RACE-Seq library. Any possible sequence at positions +2 to +8 is covered at least 100 times, but typically thousands of times during sequencing. The promoter strength of a given promoter sequence was assessed based on the relative production of transcripts from said promoter.

Generation of 5’ RACE-Seq libraries

10 ng of dsDNA template (T7_15N; SEQ ID NO:2) containing a T7 RNA polymerase promoter randomized from position +2 to +16 (gBlocks Gene Fragments, Integrated DNA Technologies) was used as input for in vitro transcription using the HiScribe T7 RNA Synthesis Kit (NEB E2040S, E2070S). After 2h incubation, RNA was purified with 1.6 volumes RNAClean XP beads (Beckman Coulter), and residual template DNA digested using the TURBO DNA- freeTM Kit (Invitrogen AMI 907). Final RNA yield was assayed using the QubitTM RNA Assay Kit. Next, 500 ng of RNA was reverse transcribed using 6.66 U/pl Superscript™ IV (Invitrogen 18090010) and 1.2 mM RT oligonucleotide. Following RNAseA and RNAseH treatment, cDNA was purified using 1.6 volumes Ampure XP beads and eluted in 10 pi water. A poly (A)-tailing reaction was carried out using 3 U/mI terminal transferase (Invitrogen 10533). Second-strand cDNA synthesis was performed in 50 pi with 0.5 pM Oligo i5_5N_dT20VN, 2000 U Q5® High- Fidelity DNA Polymerase, and 0.2 mM dNTPs, using the temperature cycle 98 °C for 30 sec; 55 °C for 30 sec; 72 °C for 5 min. Next, 2 pL Exonuclease 1 and 48 pi water were added and the reaction was incubated for 1 h at 37 °C. Then, dsDNA was purified with 1.6 volumes AMPure XP beads, and eluted in 22 pi water. Sequencing adapters were introduced during 8 cycles of PCR with KAPA HiFi HotStart. Libraries were purified twice with 1.2 volumes AMPure XP beads and sequenced using a NextSeq500 instrument from Illumina in paired-end mode (2x35), detecting the randomized 15mer sequence in Read 1 and a 5 base UMI (unique molecular identifier) in Read 2. As the adapter for second strand synthesis following polyadenylation contained a UMI, any bias introduced during the final 8 cycles of PCR could be filtered out.

For background library preparation, a total of 2.5 ng of dsDNA template (T7 15N) was amplified by PCR using KAPA HiFi HotStart Polymerase, P5 and BG T7 sequencing adapters. Here, simply 7 cycles of PCR had to be performed. Libraries were purified twice with 0.9 volumes AMPure XP beads and sequenced in single end mode using a NextSeq 500 instrument from Illumina. The first 15 nucleotides were used for data analysis.

The dsDNA template for RACE-Seq sequence was as follows (SEQ ID NO:2):

ACGAGAGCTTGTCTGCTCCCAGGCATCGCTTACAGACCTATATGACCACGCGTAT

CGATGTCGACCTACACGACGCTCTTCCGATCTTTTCATCGGGCCGTGCAGGCE44r

A CGA CTCA CTA rAGNNNNNNNNNNNNNNNCCTCTCGTTCTGTCGAAGGGC ATCGA

CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG

CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT

CAAGATCCGCCACAACATCGAGGACGCAAGATCGGAAGAGCGGTTCAGCACC

GCATCGAG wherein bold and italics refer to the T7 core sequence, bold and not in italics to the sequencing adapter binding site, and bold and underlined to the reverse transcription binding site, and wherein N denotes any nucleotide selected from the group consisting of A, C, G, and T.

Sequence data analysis

Raw reads from 5’RACE-Seq libraries were filtered for PhiX using Bowtie 2 (http://bowtie- bio.sourceforge.net/bowtie2/index.shtml). Duplicate 15mer-UMl combinations were removed. Forward reads harboring 15mer sequences were selected for the presence of a C at position 16, followed by TT, CT or CC at position 17 and 18 (to accommodate for Cs added by the reverse transcriptase to the end of cDNAs, as well as for T7 polymerase sliding in promoter variants with transcription starting on triple G sequences), followed by a polyT sequence, wherein positions refer to positions within the respective forward read. After reverse-complementing the 15mer, lOmers corresponding to position +2 to +11 in the in vitro transcribed RNA (as numbered within the T7 promoter) were mapped against the constant sequences of the sequencing library and T7 template DNA using Bowtie 2 allowing one mismatch, to remove spurious contaminating sequences. After filtering, 35c10^L6 and 84x10^L6 reads were used for motif analysis in replicates A and B, respectively. For background libraries, reads were filtered for PhiX using Bowtie 2, resulting in 232c10^L6 total reads for motif analysis. To determine relative abundances of +2 to +8 promoter variants, reads with identical +2 to +8 sequence were pooled, and the resulting read counts were divided by the total number of filtered reads and by the corresponding counts in background libraries. Homopolymeric motifs were removed from the analysis as G nucleotides appeared to be slightly disproportionally removed by the real time analysis (RTA) software application of the Illumina NextSeq 500 sequencer (reads containing stretches of dark sequencing cycles correspond to Gs in this two-fluorescence channel sequencing system).

Results of 5’RACE-Seq with T7 promoter variants and a T7 RNA polymerase

The most enriched nucleotide dimer at positions +2 and +3 consisted of two deoxyguanosines (Figure 2). Thus, in combination with the conserved deoxyguanosine at position +1, 5’RACE- Seq revealed that the most effective bases at positions +1 to +3 of the T7 promoter were three guanines (“GGG”).

A substantial sequence preference was observed within the first 8 nucleotides (+1 to +8), as shown by the high variance in the base frequency. In contrast, a comparatively low variance was observed at positions +9 to +16 (Figure 3). Hence, 5’RACE-Seq revealed that the T7 promoter strength depended primarily on the nucleotides at positions +1 to +8. The measured self-transcription activity of individual T7 promoter variants with different +4 to +8 nucleotide sequences following GGG at positions +1 to +3 was highly reproducible in two replicate experiments (Figure 4).

A comprehensive list of 5’RACE-Seq data for T7 promoter variants comprising three guanines at positions +1 to +3 and a certain set of nucleotide sequences at positions +4 to +8 is shown in Table 1. The downstream flanking regions (positions +1 to +8) of T7 promoter variants are ranked based on the transcriptional activity measured by 5’RACE-Seq. Furthermore, the normalized activity measurements of two replicate experiments (A and B) and the mean of the normalized activity obtained in the two experiments is shown.

Table 1. 5’ RACE- Seq data with T7 promoter variants

Rank Pos. +1 to +8 Activity (A) Activity (B) Mean Activity

1 GGG A A AT A 3.525168248 3.823209146 3.674188697

2 GGGAAAAT 3.438692553 3.789168012 3.613930283

3 GGG A AT AT 3.232736283 3.601622929 3.417179606 GGGATAAT 3.097185327 3.594921408 3.346053368

GGGAGAAT 3.075681634 3.586431153 3.331056393

GGGAATAC 3.053363516 3.415617863 3.23449069

GGGAAGTA 2.972851942 3.388513435 3.180682688

GGGAGATT 2.869634955 3.420750535 3.145192745

GGGAGATA 2.900774601 3.374546127 3.137660364

GGGAAATG 2.945055434 3.315268421 3.130161927

GGGAAAAC 2.924415033 3.321505123 3.122960078

GGGAAAGT 2.951492193 3.291203249 3.121347721

GGGTAAAT 2.951959296 3.27633853 3.114148913

GGGATATT 2.897644609 3.313241754 3.105443182

GGGAATAA 2.837061301 3.314414742 3.075738022

GGGATACA 2.821467492 3.317186151 3.069326822

GGGAATTA 2.876415915 3.257396204 3.06690606

GGGAGATG 2.888913879 3.167365009 3.028139444

GGGAAATT 2.904651194 3.149765628 3.027208411

GGGATATA 2.802264185 3.246699636 3.02448191

GGGTGAAT 2.778302803 3.251270686 3.014786744

GGGAAAAG 2.740336533 3.200535702 2.970436117

GGGAAAAA 2.834863563 3.105204943 2.834863563

GGGATAAC 2.763659019 3.173558391 2.968608705

GGGATACT 2.661347668 3.226933538 2.944140603

GGGTGATG 2.795002498 3.090837867 2.942920183

GGGTAATA 2.766622429 3.115855797 2.941239113

GGGTGACG 2.885805743 2.986973135 2.936389439

GGGAAAGA 2.696791071 3.16673056 2.931760815

GGGAATAG 2.676987678 3.178098041 2.92754286

GGGTGACT 2.739784061 3.103831997 2.921808029

GGGTGACA 2.73581603 3.085354489 2.910585259 GGGAAGAT 2.640417175 3.175261328 2.907839252

GGGAGACT 2.668992818 3.143510608 2.906251713

GGGATAAG 2.693674813 3.118556314 2.906115564

GGGTGATT 2.696857823 3.114911609 2.905884716

GGGAAGTT 2.65827908 3.153233322 2.905756201

GGGTAAGT 2.740432423 3.019535979 2.879984201

GGGTAATG 2.706213926 3.049661779 2.877937852

GGGTAATT 2.718573103 3.028335092 2.873454097

GGGATATG 2.598076221 3.137393258 2.86773474

GGGAGAAA 2.631782575 3.095781911 2.863782243

GGGAAACA 2.670917539 3.050891454 2.860904497

GGGTGATA 2.615365374 3.098605087 2.85698523

GGGAAATC 2.704008581 3.007839613 2.855924097

GGGATTAT 2.616186414 3.081415351 2.848800882

GGGTAATC 2.692477936 2.991362364 2.84192015

GGGTGAAG 2.673033067 3.009394835 2.841213951

GGGATAGT 2.649694018 3.03117066 2.840432339

GGGATAAA 2.614840591 3.060912475 2.837876533

GGGATTAC 2.618533517 2.991334561 2.804934039

GGGAGACA 2.607068837 2.999938955 2.803503896

GGGAGTAT 2.522614184 3.082969421 2.802791802

GGGTGAGT 2.664084601 2.937448795 2.800766698

GGGTATAT 2.61460393 2.985719391 2.80016166

GGGAGATC 2.510935934 3.037315306 2.77412562

GGGACAAT 2.534079055 3.005570996 2.769825025

GGGAAGAC 2.573541755 2.962569118 2.768055437

GGGTAAAG 2.636775491 2.880652312 2.758713901

GGGATATC 2.556732902 2.959057729 2.757895316

GGGAAGTC 2.631055179 2.881123403 2.756089291 GGGAGAAC 2.539694252 2.941637993 2.740666122

GGGAGACG 2.543611003 2.934566036 2.73908852

GGGAAGTG 2.516972031 2.922638554 2.719805292

GGGTAGAT 2.583853931 2.854888699 2.719371315

GGGAGAGT 2.492741578 2.941430236 2.717085907

GGGAATGT 2.49929406 2.909344717 2.704319389

GGGAGAAG 2.506781408 2.899277796 2.703029602

GGGTAGTT 2.555265009 2.846707963 2.700986486

GGGTGTAG 2.615352515 2.778187954 2.696770234

GGGTAACA 2.533091616 2.848794729 2.690943172

GGGAGTAG 2.440985911 2.903625647 2.672305779

GGGAATGA 2.443050742 2.894136575 2.668593658

GGGACATA 2.415643828 2.908091944 2.661867886

GGGAAACT 2.444148231 2.875473019 2.659810625

GGGAACTA 2.430998927 2.888583778 2.659791352

GGGTGATC 2.419206738 2.894117701 2.65666222

GGGTAACT 2.444109396 2.850656605 2.647383

GGGTGTAT 2.424965377 2.856765463 2.64086542

GGGATAGA 2.467401891 2.813455435 2.640428663

GGGTATTA 2.465268736 2.79993901 2.632603873

GGGATTAG 2.350601593 2.893425139 2.622013366

GGGAATTG 2.455477307 2.759835535 2.607656421

GGGATTAA 2.398889072 2.809019347 2.60395421

GGGTAGTC 2.513950486 2.675168106 2.594559296

GGGTAGTG 2.477116173 2.711622605 2.594369389

GGGATAGC 2.351672105 2.835310639 2.593491372

GGGTAAAC 2.439773564 2.743370846 2.591572205

GGGTAGAC 2.424153979 2.745254592 2.584704286

GGGTACAT 2.43555543 2.733071621 2.584313526 GGGTAAGC 2.469103654 2.69475792 2.581930787

GGGAGTAA 2.314737543 2.823695711 2.569216627

GGGAACAC 2.323461972 2.811493817 2.567477895

GGGTGAAA 2.330090711 2.801544034 2.565817372

GGGATACG 2.320300681 2.809100901 2.564700791

GGGAAACG 2.405717439 2.707372291 2.556544865

GGGTGAGC 2.423439891 2.6876188 2.555529346

GGGTAGTA 2.394560426 2.70744816 2.551004293

GGGAAAGC 2.353424293 2.728586852 2.541005572

GGGACAAC 2.246809452 2.8266996 2.536754526

GGGAAGAA 2.26851203 2.803885432 2.536198731

GGGACATT 2.329625189 2.741597047 2.535611118

GGGTATTG 2.367929344 2.69411322 2.531021282

GGGAGTTA 2.298925226 2.747558214 2.52324172

GGGACACA 2.281647623 2.759472589 2.520560106

GGGAACAT 2.322575333 2.718004853 2.520290093

GGGTAAGA 2.337434156 2.696779147 2.517106652

GGGAATCA 2.263582631 2.746542613 2.505062622

GGGGAATA 2.264737749 2.731519678 2.498128714

GGGAACAA 2.219442057 2.774576166 2.497009112

GGGAAACC 2.291204308 2.699796118 2.495500213

GGGTAACG 2.339762518 2.649176272 2.494469395

GGGTACAC 2.340027025 2.630472091 2.485249558

GGGTCATA 2.408042629 2.553972856 2.481007743

GGGATGAT 2.259865364 2.695257376 2.47756137

GGGACAGT 2.274875643 2.661297488 2.468086565

GGGTGTAA 2.233272358 2.670932091 2.452102224

GGGTATAA 2.268812122 2.628036487 2.448424304

GGGAATGC 2.226058142 2.670720286 2.448389214 GGGTCAAT 2.321239547 2.572445229 2.446842388

GGGAACAG 2.187053649 2.704966571 2.44601011

GGGTGAGA 2.288140393 2.599166118 2.443653256

GGGTATTC 2.287414018 2.590367798 2.438890908

GGGTGTAC 2.256179165 2.609760255 2.43296971

GGGAGTAC 2.276089658 2.583595912 2.429842785

GGGTATGT 2.308667464 2.533499682 2.421083573

GGGAATCT 2.207419595 2.61344807 2.410433832

GGGACACT 2.160048721 2.653218578 2.40663365

GGGTAGGT 2.301560408 2.504926886 2.403243647

GGGTACTA 2.25242405 2.533334944 2.392879497

GGGTATAG 2.25569686 2.515430556 2.385563708

GGGACAAA 2.112463309 2.635037684 2.373750496

GGGATACC 2.15880511 2.582377789 2.370591449

GGGAGAGC 2.222711715 2.511843928 2.367277821

GGGTACAG 2.208226606 2.522376518 2.365301562

GGGACATG 2.17763587 2.548977868 2.363306869

GGGATGAC 2.186835287 2.536098983 2.361467135

GGGTGTTA 2.188037663 2.5288003 2.358418981

GGGACAAG 2.135488316 2.580642926 2.358065621

GGGTATAC 2.231101467 2.476377725 2.353739596

GGGGAAAT 2.172462515 2.534126221 2.353294368

GGGAGAGA 2.154888899 2.545266441 2.35007767

GGGGAATT 2.133100297 2.545183507 2.339141902

GGGTACAA 2.130819175 2.546468329 2.338643752

GGGTATCA 2.159975668 2.516627535 2.338301601

GGGAGACC 2.030706645 2.633735328 2.332220986

GGGTGAAC 2.216872909 2.447407227 2.332140068

GGGATTTA 2.130172498 2.532604807 2.331388652 GGGTAGAA 2.148878507 2.513485388 2.331181948

GGGGATTA 2.141310831 2.510496704 2.325903768

GGGAATTT 2.18858784 2.461124006 2.324855923

GGGTGTGT 2.198335029 2.444737988 2.321536508

GGGTGTCT 2.174406603 2.457650545 2.316028574

GGGAACCA 2.056753591 2.569208373 2.312980982

GGGAGTTG 2.144283404 2.480395815 2.312339609

GGGTGACC 2.070818868 2.515940589 2.293379728

GGGTATGA 2.116762323 2.44124428 2.279003302

GGGACAGA 2.112343544 2.444615914 2.278479729

GGGAAAGG 2.086520294 2.465835554 2.276177924

GGGATTGA 2.07577825 2.46933734 2.272557795

GGGAATGG 2.057805742 2.480679717 2.26924273

GGGAATTC 2.089684697 2.448179526 2.268932111

GGGTACTG 2.136828571 2.400067079 2.268447825

GGGAACTC 1.990947214 2.545818409 2.268382812

GGGTGTCA 2.132187157 2.396965306 2.264576231

GGGACACG 2.040543386 2.482632121 2.261587754

GGGTATTT 2.124420198 2.390429513 2.257424856

GGGGATAT 2.061329679 2.452278288 2.256803983

GGGACATC 2.012648332 2.487042106 2.249845219

GGGACTAT 2.036250084 2.462174887 2.249212485

GGGAATCC 2.004999389 2.488090451 2.24654492

GGGTTAAT 2.108810747 2.382916154 2.245863451

GGGTAAAA 2.114827135 2.373009687 2.243918411

GGGTAACC 2.073748571 2.406702831 2.240225701

GGGATGTA 2.064964503 2.406583709 2.235774106

GGGTGTCG 2.194064494 2.273300293 2.233682393

GGGAGTTC 2.029853974 2.431526858 2.230690416 GGGTACTT 2.051229964 2.407713149 2.229471557

GGGGAATC 2.001475686 2.456648719 2.229062202

GGGGAATG 2.039064528 2.415949439 2.227506983

GGGACAGC 2.058922725 2.386210877 2.222566801

GGGAACTT 1.982161271 2.450320651 2.216240961

GGGACTAC 2.001614949 2.415561718 2.208588334

GGGTGTTC 2.007655259 2.397526441 2.20259085

GGGTACTC 2.10743426 2.284449499 2.19594188

GGGAGTGT 1.962581201 2.409956088 2.186268644

GGGAGTCC 2.001160528 2.342501865 2.171831197

GGGATGAA 1.98644248 2.355115159 2.170778819

GGGAACCT 1.978036933 2.361030766 2.16953385

GGGTGAGG 2.049138883 2.270687567 2.159913225

GGGAGTGG 1.98084729 2.332774235 2.156810762

GGGTTATT 2.003451267 2.304575258 2.154013263

GGGTATGC 2.030888693 2.275129093 2.153008893

GGGAACTG 1.919623052 2.37333938 2.146481216

GGGATGAG 1.978952839 2.306845553 2.142899196

GGGTTACA 2.005904056 2.277918985 2.14191152

GGGAGTGA 1.923839752 2.35691958 2.140379666

GGGTTATG 2.033259992 2.245053466 2.139156729

GGGTGTGA 1.973488452 2.299700215 2.136594333

GGGTCATT 2.018069144 2.254047135 2.136058139

GGGGATTG 1.986976034 2.270625643 2.128800839

GGGTGTGC 1.986333191 2.26705449 2.12669384

GGGATTGT 1.974482657 2.265271634 2.119877145

GGGTATCT 1.923758953 2.313401648 2.118580301

GGGTAGAG 2.000314625 2.234481254 2.11739794

GGGAGTCT 1.933203058 2.289694262 2.11144866 GGGAAGCC 1.951507181 2.269882841 2.110695011

GGGTTAAG 1.961308247 2.258714887 2.110011567

GGGATTCA 1.918470215 2.300013379 2.109241797

GGGAGTGC 1.928866039 2.283330454 2.106098246

GGGTTATA 1.947486113 2.26231139 2.104898752

GGGTACGA 1.949726554 2.245653816 2.097690185

GGGTACGT 1.994707803 2.196961156 2.09583448

GGGTATCG 1.928579924 2.23642074 2.082500332

GGGTGTGG 1.978837818 2.183286129 2.081061973

GGGGATAC 1.897417132 2.263868607 2.08064287

GGGATAGG 1.900245625 2.260756457 2.080501041

GGGTTACT 1.917138265 2.233515628 2.075326946

GGGATGTG 1.908908946 2.238603734 2.07375634

GGGGATTC 1.884724439 2.256374458 2.070549449

GGGAGTCA 1.88628317 2.243065618 2.064674394

GGGTAGCC 1.919484828 2.190115597 2.054800213

GGGTCAAG 1.879268387 2.218725531 2.048996959

GGGTGTTG 1.884623073 2.205325625 2.044974349

GGGACTAA 1.86597052 2.219800333 2.042885427

GGGGAACT 1.859803904 2.220456335 2.04013012

GGGGATAA 1.863240128 2.211652305 2.037446216

GGGTCACA 1.872957221 2.1967677 2.03486246

GGGGAAAG 1.885389902 2.178418754 2.031904328

GGGATCAT 1.776386418 2.282007784 2.029197101

GGGAATCG 1.851352612 2.204271098 2.027811855

GGGGTAAT 1.856054509 2.195058451 2.02555648

GGGATTGG 1.833951997 2.203394501 2.018673249

GGGTGTTT 1.822871343 2.213474158 2.01817275

GGGGAAAC 1.85390056 2.178736954 2.016318757 GGGAACGA 1.811149881 2.217627235 2.014388558

GGGAGTTT 1.818170659 2.208710908 2.013440783

GGGCAGAT 1.801456648 2.222732349 2.012094498

GGGTTAGT 1.891836656 2.12998131 2.010908983

GGGTCAAA 1.845984922 2.15868546 2.002335191

GGGAAGGT 1.825490677 2.174186025 1.999838351

GGGATTTG 1.833596725 2.163511681 1.998554203

GGGGAAGT 1.814243726 2.179295865 1.996769795

GGGTCAAC 1.842827604 2.140469817 1.99164871

GGGTTACG 1.836256406 2.146983736 1.991620071

GGGATTCT 1.804700296 2.177155428 1.990927862

GGGTTAAA 1.853838743 2.126031942 1.989935343

GGGGATAG 1.846680125 2.132792 1.989736063

GGGACTAG 1.809495949 2.16648971 1.98799283

GGGATTGC 1.787847117 2.187882494 1.987864806

GGGAACGT 1.779509345 2.194668607 1.987088976

GGGCAAAT 1.769460291 2.197696402 1.983578346

GGGCAATA 1.736199936 2.222101206 1.979150571

GGGTAAGG 1.807098143 2.13709442 1.972096281

GGGGACAT 1.779846652 2.161025476 1.970436064

GGGTATCC 1.779402162 2.138804277 1.959103219

GGGATGTC 1.807447869 2.109060772 1.95825432

GGGGATCA 1.808455369 2.103912761 1.956184065

GGGTTAAC 1.843988526 2.068056711 1.956022618

GGGTTATC 1.803601166 2.105858303 1.954729734

GGGGTATT 1.786264531 2.117142725 1.951703628

GGGGTACA 1.794289886 2.10848321 1.951386548

GGGCAGAC 1.730448272 2.171266719 1.950857495

GGGAGTCG 1.838655383 2.062612176 1.950633779 GGGGTACT 1.801854803 2.092110181 1.946982492

GGGACACC 1.77132266 2.121212474 1.946267567

GGGTAGCT 1.769324462 2.109812526 1.939568494

GGGATCAG 1.713688598 2.163470211 1.938579404

GGGGAACA 1.757411931 2.117862017 1.937636974

GGGTCAGT 1.837620369 2.031553479 1.934586924

GGGTACGC 1.715353221 2.147611128 1.931482174

GGGGACTA 1.775581273 2.086763029 1.931172151

GGGTTAGA 1.786527721 2.072526492 1.929527106

GGGTCATG 1.845253936 1.992488922 1.918871429

GGGTGGAT 1.752532486 2.077762209 1.915147348

GGGTAGCA 1.762845665 2.057330281 1.910087973

GGGTGTCC 1.725938302 2.083058753 1.904498528

GGGACAGG 1.686501109 2.122330168 1.904415638

GGGATCAA 1.672341435 2.132516982 1.902429209

GGGTACCA 1.7406267 2.062709357 1.901668029

GGGCAAAC 1.661357046 2.137396145 1.899376596

GGGGAACG 1.749084075 2.04670937 1.897896722

GGGGTAAC 1.777022003 2.016881658 1.896951831

GGGGACAA 1.693414331 2.099698345 1.896556338

GGGATCAC 1.635619135 2.150292911 1.892956023

GGGGACAG 1.733449263 2.048787741 1.891118502

GGGTAGCG 1.776748302 2.001880584 1.889314443

GGGTAGGC 1.793314381 1.982093153 1.887703767

GGGCATAT 1.63624506 2.126471303 1.881358182

GGGAAGAG 1.73378526 2.026863781 1.880324521

GGGACTTA 1.693202305 2.063148682 1.878175494

GGGTTAGC 1.74071129 2.015219347 1.877965318

GGGCGAAT 1.669320912 2.066414686 1.867867799 GGGTCACG 1.725618522 2.002765097 1.86419181

GGGAACGC 1.670765666 2.057238826 1.864002246

GGGTATGG 1.694779359 2.029463821 1.86212159

GGGATTTC 1.707513263 2.006913807 1.857213535

GGGGATTT 1.669204112 2.036490769 1.85284744

GGGGTAGT 1.754650412 1.950374087 1.85251225

GGGATTCG 1.692832615 2.010368803 1.851600709

GGGTCACT 1.683783529 2.017823831 1.85080368

GGGCAATG 1.625698706 2.073594707 1.849646706

GGGGTATA 1.670594464 2.01302099 1.841807727

GGGGTAAG 1.708796353 1.968739394 1.838767874

GGGCAAGT 1.64458869 2.027414308 1.836001499

GGGAAGGA 1.690504412 1.980296687 1.835400549

GGGGTATG 1.686322928 1.974051142 1.830187035

GGGGTTAT 1.62646437 2.03177573 1.82912005

GGGGTACG 1.688328901 1.966081093 1.827204997

GGGATTCC 1.638941658 2.007231792 1.823086725

GGGGAAAA 1.672829066 1.966547355 1.81968821

GGGTGGTT 1.593443902 2.023827303 1.808635602

GGGACTCA 1.648432061 1.96549606 1.80696406

GGGTTGAT 1.662083756 1.947197737 1.804640746

GGGTGGAC 1.613064753 1.995787984 1.804426368

GGGGATGT 1.662265782 1.943765994 1.803015888

GGGATCTA 1.583372966 2.021305925 1.802339446

GGGGATGA 1.70372902 1.897891153 1.800810087

GGGCAAAG 1.570885798 2.029499293 1.800192546

GGGTCATC 1.688791496 1.908520868 1.798656182

GGGCAGTA 1.573127458 2.016218327 1.794672892

GGGAACCC 1.593360743 1.994660137 1.79401044 GGGGATCT 1.5841111 2.000569639 1.79234037

GGGGAGTT 1.595272827 1.987373176 1.791323001

GGGGTATC 1.642562982 1.939046191 1.790804587

GGGCAATC 1.526138714 2.054371814 1.790255264

GGGGATGC 1.667925593 1.901832917 1.784879255

GGGATGTT 1.624490234 1.944619888 1.784555061

GGGGTTAC 1.597995144 1.970107387 1.784051266

GGGTCAGA 1.655173077 1.90908927 1.782131173

GGGATGGC 1.61298302 1.948734118 1.780858569

GGGGACTG 1.605119159 1.943975019 1.774547089

GGGTGGTA 1.617700962 1.93080561 1.774253286

GGGTTGAG 1.679437455 1.865515321 1.772476388

GGGGTAAA 1.63252583 1.906202708 1.769364269

GGGGACTC 1.630680544 1.907510477 1.769095511

GGGACTGA 1.550643585 1.979550997 1.765097291

GGGGACAC 1.592349826 1.917966732 1.755158279

GGGTCAGC 1.656889813 1.848468341 1.752679077

GGGTCTAT 1.609816684 1.878214127 1.744015405

GGGTGGAG 1.630971685 1.856199831 1.743585758

GGGGATCG 1.60261806 1.881831213 1.742224637

GGGAACCG 1.556661433 1.918884348 1.73777289

GGGGACTT 1.532038828 1.943054043 1.737546436

GGGAACGG 1.553036029 1.919142579 1.736089304

GGGGAGTA 1.554869343 1.912314022 1.733591682

GGGCAGTC 1.52701458 1.940032102 1.733523341

GGGCTAAC 1.511457346 1.947263378 1.729360362

GGGCAGTG 1.526367853 1.90433955 1.715353702

GGGGTTAG 1.525572092 1.904637137 1.715104615

GGGCAATT 1.470997857 1.95474127 1.712869564 GGGATGGA 1.563489467 1.855498032 1.709493749

GGGTAGGA 1.583340067 1.834319804 1.708829936

GGGCGAAC 1.429720572 1.986701602 1.708211087

GGGAGAGG 1.575359746 1.839136342 1.707248044

GGGGAGTC 1.555059557 1.857268326 1.706163941

GGGCAAGA 1.522289184 1.887797475 1.705043329

GGGAAGCA 1.528104647 1.875894815 1.701999731

GGGATGCC 1.580459643 1.821511285 1.700985464

GGGGATCC 1.515186554 1.885826632 1.700506593

GGGTTTAT 1.545238609 1.854616224 1.699927417

GGGCGATA 1.460915718 1.93571387 1.698314794

GGGGTTAA 1.5073654 1.880395033 1.693880216

GGGACTGT 1.506244801 1.877306662 1.691775731

GGGTGGTC 1.609705307 1.773005387 1.691355347

GGGACTCT 1.514610124 1.866191758 1.690400941

GGGATGGT 1.514898672 1.865058901 1.689978786

GGGTGGAA 1.516743556 1.856077199 1.686410378

GGGTACCT 1.543940408 1.820318887 1.682129648

GGGTGGTG 1.535761259 1.826622504 1.681191882

GGGGAAGA 1.478138965 1.877285853 1.677712409

GGGATGCT 1.456603492 1.8890316 1.672817546

GGGAGGAT 1.494080791 1.847644161 1.670862476

GGGACTTG 1.510541129 1.821692682 1.666116905

GGGTGGCG 1.645349808 1.683769409 1.664559608

GGGCAGTT 1.439173432 1.882698028 1.66093573

GGGATCCA 1.469075831 1.84711568 1.658095755

GGGCGATG 1.472830846 1.842379724 1.657605285

GGGCGAAG 1.457935462 1.853300753 1.655618107

GGGTCTAA 1.482885656 1.823722267 1.653303961 GGGCTACT 1.463545566 1.841685823 1.652615695

GGGAAGCT 1.486008897 1.816850091 1.651429494

GGGTCTAC 1.544931566 1.748979523 1.646955545

GGGACTCG 1.520591474 1.773267005 1.646929239

GGGCAGAG 1.471302937 1.820186931 1.645744934

GGGTACGG 1.487768002 1.798865575 1.643316788

GGGCCATT 1.481861123 1.803900752 1.642880938

GGGCTACA 1.461861958 1.821151072 1.641506515

GGGCCAAT 1.395127211 1.886170583 1.640648897

GGGCATAG 1.400317969 1.876220267 1.638269118

GGGAGCCA 1.477066272 1.793441079 1.635253675

GGGCTAAG 1.431618202 1.838726933 1.635172567

GGGAAGGC 1.454240347 1.813098852 1.6336696

GGGGAAGC 1.489272707 1.776972389 1.633122548

GGGGTAGC 1.506115066 1.759395398 1.632755232

GGGATGCA 1.447850443 1.802776458 1.625313451

GGGTTTAA 1.466354467 1.783656194 1.62500533

GGGTTTAG 1.489736855 1.760137868 1.624937361

GGGGTTGT 1.49651839 1.748225411 1.622371901

GGGCAAGG 1.424995544 1.81876264 1.621879092

GGGCCACA 1.436218502 1.806971468 1.621594985

GGGTTACC 1.484405655 1.757551991 1.620978823

GGGACTGC 1.482441838 1.756748767 1.619595302

GGGTTGAC 1.4835237 1.751966903 1.617745302

GGGACCAT 1.391563902 1.843761574 1.617662738

GGGCTATT 1.389697114 1.844599631 1.617148372

GGGACGAC 1.471281341 1.753290773 1.612286057

GGGGAGTG 1.50063389 1.719468583 1.610051236

GGGCTATC 1.38764757 1.828850422 1.608248996 GGGCAACC 1.358050489 1.856238652 1.607144571

GGGTTAGG 1.4717753 1.740323148 1.606049224

GGGTTGAA 1.47087015 1.735764665 1.603317408

GGGCCAAC 1.436699332 1.76916963 1.602934481

GGGGAGAT 1.402145235 1.802664535 1.602404885

GGGCAGAA 1.41410585 1.790127941 1.602116896

GGGTGGCT 1.513493212 1.686508095 1.600000654

GGGCGACT 1.312354396 1.887007343 1.59968087

GGGCCATA 1.382569396 1.815520858 1.599045127

GGGGAACC 1.380503305 1.816507548 1.598505427

GGGAGGTG 1.457080956 1.732427301 1.594754128

GGGTCAGG 1.497351152 1.691501026 1.594426089

GGGAGGTA 1.441960796 1.746654735 1.594307766

GGGCTTAT 1.392255014 1.795844272 1.594049643

GGGCCAGT 1.43039275 1.754314482 1.592353616

GGGCAACT 1.373333695 1.804899146 1.589116421

GGGCATAC 1.377498049 1.800576227 1.589037138

GGGGTTGA 1.433340241 1.744351841 1.588846041

GGGACGAT 1.394853751 1.779439992 1.587146872

GGGACCAC 1.366687654 1.806889838 1.586788746

GGGCAAGC 1.352731406 1.820770559 1.586750982

GGGACTTC 1.403547037 1.766693147 1.585120092

GGGCGACA 1.359292129 1.805348834 1.582320482

GGGAGCCT 1.393461798 1.765522529 1.579492164

GGGGTAGA 1.460561415 1.692538316 1.576549866

GGGCTACG 1.404321615 1.747978846 1.57615023

GGGCACAC 1.386118947 1.764020613 1.57506978

GGGTTGTG 1.522599995 1.621944101 1.572272048

GGGCTATA 1.377250619 1.763506856 1.570378738 GGGACCAG 1.342424906 1.797054914 1.56973991

GGGCCACT 1.377106659 1.756823596 1.566965127

GGGCTAAT 1.3904259 1.741354544 1.565890222

GGGCATAA 1.372483573 1.759021256 1.565752415

GGGCGTAT 1.40132169 1.729406043 1.565363867

GGGACTGG 1.420247216 1.710235376 1.565241296

GGGCACAT 1.371738926 1.755168094 1.56345351

GGGCTATG 1.381549424 1.740212673 1.560881048

GGGGAGAC 1.376454908 1.744805575 1.560630242

GGGCAACG 1.382860748 1.736910151 1.55988545

GGGTCTCA 1.436485865 1.682769966 1.559627916

GGGTCTAG 1.424928646 1.688187817 1.556558231

GGGCCAGC 1.385405381 1.722791147 1.554098264

GGGCATTA 1.346556974 1.758832655 1.552694814

GGGATGCG 1.35726161 1.747988087 1.552624848

GGGAGCAT 1.36925603 1.732111841 1.550683935

GGGCGATT 1.310772016 1.788839949 1.549805982

GGGCAGGT 1.347839216 1.747456287 1.547647751

GGGCGAGT 1.338167127 1.756998151 1.547582639

GGGAGCCC 1.427378836 1.658784861 1.543081849

GGGAGCAA 1.39261322 1.686631705 1.539622462

GGGATCTG 1.314061706 1.764628505 1.539345106

GGGCCAAG 1.336849487 1.741391858 1.539120673

GGGCTAGT 1.360885996 1.714869427 1.537877712

GGGCACAG 1.329583517 1.732671993 1.531127755

GGGAGGAA 1.364904501 1.694282761 1.529593631

GGGAGGAG 1.426232735 1.632894288 1.529563512

GGGAAGCG 1.37697587 1.678792607 1.527884239

GGGGACGT 1.41293951 1.640203505 1.526571508 GGGACTCC 1.399591498 1.649627215 1.524609357

GGGCTAGA 1.339816238 1.702635776 1.521226007

GGGCGATC 1.235044686 1.801303449 1.518174068

GGGTGGCA 1.412128433 1.623400169 1.517764301

GGGCGACG 1.33013329 1.704435473 1.517284382

GGGAGGTT 1.316687896 1.717068602 1.516878249

GGGATCCT 1.304687068 1.726814863 1.515750965

GGGCTGAC 1.339572808 1.690562329 1.515067569

GGGGCAAT 1.331998275 1.697109582 1.514553929

GGGCAACA 1.332695462 1.696025918 1.51436069

GGGTTTAC 1.376185845 1.64734778 1.511766812

GGGATCTC 1.315294526 1.70650872 1.510901623

GGGGTGAT 1.347381879 1.674260883 1.510821381

GGGGCAAG 1.334151845 1.685965897 1.510058871

GGGACCAA 1.311266617 1.705467719 1.508367168

GGGATCCG 1.291676679 1.721112137 1.506394408

GGGACTTT 1.356008382 1.656083368 1.506045875

GGGCAGGA 1.332176668 1.67846716 1.505321914

GGGAGGTC 1.398761864 1.609112555 1.503937209

GGGGTTTA 1.306299152 1.700034139 1.503166645

GGGCTAAA 1.306639007 1.696022282 1.501330645

GGGTTGTT 1.385544755 1.616691808 1.501118282

GGGACGTA 1.35863433 1.641221166 1.499927748

GGGGTGAC 1.377753902 1.61836542 1.498059661

GGGCCACG 1.322506508 1.663946091 1.493226299

GGGAGCCG 1.360848844 1.621678261 1.491263552

GGGCCAGA 1.291231679 1.690790453 1.491011066

GGGCTTAC 1.263874216 1.717338556 1.490606386

GGGTGCCT 1.324572799 1.656554119 1.490563459 GGGTACCG 1.313317613 1.653491277 1.483404445

GGGCAGCC 1.290079787 1.674263221 1.482171504

GGGCGTAC 1.290621825 1.670012963 1.480317394

GGGGAGAA 1.279350807 1.673343769 1.476347288

GGGGATGG 1.306002938 1.644952449 1.475477694

GGGCTACC 1.263986157 1.685677626 1.474831892

GGGCTTAG 1.291760993 1.654991885 1.473376439

GGGTCTTA 1.343713634 1.601447539 1.472580587

GGGCGGAC 1.416702128 1.517736966 1.467219547

GGGGTTCT 1.25183187 1.661183914 1.456507892

GGGGTTGG 1.321329655 1.589928483 1.455629069

GGGTTGTA 1.37666463 1.531249939 1.453957284

GGGTCTGA 1.353428072 1.553499661 1.453463867

GGGTGCCA 1.324037826 1.582243247 1.453140537

GGGGTTCA 1.241196334 1.659907262 1.450551798

GGGCAGCA 1.27745916 1.621221239 1.4493402

GGGCGAGG 1.288401165 1.609981022 1.449191093

GGGCATGT 1.25216033 1.641891285 1.447025808

GGGGTGTA 1.286890826 1.605329169 1.446109998

GGGGTCAT 1.316313378 1.5743067 1.445310039

GGGTCTCT 1.293983651 1.596126943 1.445055297

GGGTGGGC 1.287293885 1.599271809 1.443282847

GGGCATGA 1.293992061 1.591382317 1.442687189

GGGATCGA 1.25756324 1.621385426 1.439474333

GGGGACGA 1.296999648 1.57763962 1.437319634

GGGCGAAA 1.251814471 1.622814201 1.437314336

GGGTGGCC 1.300467157 1.570149528 1.435308343

GGGCATTG 1.249706621 1.618909159 1.43430789

GGGCATTC 1.218023868 1.65013139 1.434077629 GGGTCACC 1.275117798 1.588447256 1.431782527

GGGATCGT 1.264167937 1.597533527 1.430850732

GGGCTGAA 1.328116834 1.52346481 1.425790822

GGGTGCAA 1.260561764 1.589299505 1.424930635

GGGAGCTA 1.274231748 1.572575147 1.423403447

GGGGCATA 1.227951781 1.614338972 1.421145377

GGGCGAGC 1.233813092 1.605141516 1.419477304

GGGCCATC 1.198043039 1.638488997 1.418266018

GGGAGGAC 1.222623699 1.613529124 1.418076411

GGGAGCAG 1.325774538 1.506345834 1.416060186

GGGACCTC 1.222005738 1.609548825 1.415777282

GGGCGAGA 1.31076846 1.518002703 1.414385581

GGGACGAA 1.256057541 1.563288613 1.409673077

GGGGTTGC 1.271879776 1.546688261 1.409284019

GGGCGTAG 1.231711271 1.581772656 1.406741964

GGGCACTA 1.200263293 1.612695098 1.406479196

GGGACCTA 1.215693453 1.587225511 1.401459482

GGGTTTTA 1.282186645 1.516913458 1.399550051

GGGCCAAA 1.212886068 1.586176229 1.399531148

GGGACGTG 1.246400013 1.550856349 1.398628181

GGGGCAGA 1.286640703 1.499536328 1.393088516

GGGCACCA 1.185204245 1.596761683 1.390982964

GGGGAAGG 1.266922378 1.50419376 1.385558069

GGGGTGAA 1.205101189 1.555804405 1.380452797

GGGCAGCG 1.222680785 1.534712746 1.378696765

GGGGCAAC 1.174514181 1.576266764 1.375390472

GGGGTTTG 1.225639141 1.52102262 1.373330881

GGGTGCAT 1.20836358 1.533312312 1.370837946

GGGACGTT 1.196648275 1.544411956 1.370530115 GGGCGTAA 1.214565666 1.520253456 1.367409561

GGGGCATG 1.228579918 1.497883539 1.363231729

GGGGACGC 1.251894862 1.469558775 1.360726819

GGGGACCA 1.17974468 1.53922331 1.359483995

GGGGCACG 1.207036721 1.506721657 1.356879189

GGGGCAGT 1.176893459 1.53527039 1.356081925

GGGCAGCT 1.166147951 1.54373288 1.354940415

GGGCTTAA 1.172425555 1.53424909 1.353337322

GGGTTTGA 1.242218594 1.463802047 1.35301032

GGGGTTCG 1.165947905 1.539375975 1.35266194

GGGATCGC 1.161386807 1.539014024 1.350200415

GGGGTGTG 1.247298863 1.452310299 1.349804581

GGGCACTG 1.138470376 1.55100549 1.344737933

GGGCACTC 1.167060504 1.517037521 1.342049013

GGGCTAGC 1.16877609 1.507389291 1.33808269

GGGGTAGG 1.23564343 1.436594587 1.336119009

GGGGCATT 1.155021679 1.516843086 1.335932383

GGGTGCTA 1.168990621 1.501651876 1.335321249

GGGAGCAC 1.229726213 1.440480614 1.335103413

GGGATCGG 1.154638495 1.508409985 1.33152424

GGGCTAGG 1.162104612 1.497646619 1.329875615

GGGCTGAT 1.123587938 1.53489989 1.329243914

GGGTTTCA 1.185280615 1.470745594 1.328013105

GGGCCATG 1.183884642 1.472084329 1.327984485

GGGTTCAT 1.174715658 1.479440281 1.32707797

GGGACGGA 1.239702773 1.410834089 1.325268431

GGGGTGAG 1.194933987 1.453054235 1.323994111

GGGGTACC 1.187140147 1.460733306 1.323936727

GGGACCTT 1.153461132 1.494390687 1.32392591 GGGCATGG 1.141956823 1.503604352 1.322780587

GGGCATCC 1.116970107 1.528520049 1.322745078

GGGTTGGT 1.213708867 1.42561064 1.319659753

GGGTTGGA 1.206566406 1.432154998 1.319360702

GGGACGAG 1.129966018 1.508739384 1.319352701

GGGTGCTT 1.153617836 1.483917978 1.318767907

GGGCCAGG 1.149586096 1.48770152 1.318643808

GGGGCTAT 1.127499196 1.508667514 1.318083355

GGGCCTAC 1.172135181 1.461417432 1.316776307

GGGCAGGC 1.136149706 1.49662025 1.316384978

GGGAGCTG 1.219789571 1.412709304 1.316249438

GGGCATTT 1.127315327 1.505117692 1.316216509

GGGCATGC 1.141866436 1.489336536 1.315601486

GGGCTTTA 1.119950501 1.509177047 1.314563774

GGGGTGGC 1.233684417 1.394546834 1.314115626

GGGGCAAA 1.11343278 1.513768491 1.313600636

GGGCTGAG 1.148972598 1.477731372 1.313351985

GGGATCTT 1.118424888 1.507097982 1.312761435

GGGTGCAG 1.18485031 1.436784829 1.31081757

GGGGCTAC 1.15126594 1.467586369 1.309426155

GGGGACGG 1.269003395 1.349686897 1.309345146

GGGGCTAG 1.164638464 1.451964248 1.308301356

GGGTGGGT 1.162275835 1.449291071 1.305783453

GGGACCTG 1.139378532 1.472135584 1.305757058

GGGCAAAA 1.087953978 1.51853905 1.303246514

GGGAGCTT 1.151827059 1.454253451 1.303040255

GGGGACCT 1.09811049 1.506845146 1.302477818

GGGGTGTT 1.150050571 1.447013382 1.298531977

GGGTGCCG 1.180511478 1.41426225 1.297386864 GGGTCTCG 1.215036189 1.376562749 1.295799469

GGGGTGTC 1.189361571 1.400644946 1.295003259

GGGGAGCC 1.144723879 1.44488174 1.294802809

GGGCCACC 1.098979907 1.487988671 1.293484289

GGGTTGGC 1.185522406 1.398577526 1.292049966

GGGTTCAA 1.162783994 1.420219302 1.291501648

GGGGTCAA 1.113394585 1.468354689 1.290874637

GGGCACGG 1.126094726 1.454136159 1.290115443

GGGGTTCC 1.084441997 1.49024448 1.287343238

GGGCACGT 1.093257479 1.477056378 1.285156929

GGGGCAGC 1.147364171 1.413810081 1.280587126

GGGTTTCT 1.141472283 1.419286836 1.280379559

GGGCATCT 1.082059481 1.473159701 1.277609591

GGGATGGG 1.142405913 1.411396875 1.276901394

GGGTCGAG 1.171565411 1.380604425 1.276084918

GGGCACGC 1.053120266 1.498806231 1.275963249

GGGCCTAT 1.089637635 1.458613603 1.274125619

GGGTCGAA 1.135912262 1.407487061 1.271699662

GGGATTTT 1.158342091 1.382679144 1.270510617

GGGGTCAG 1.14229633 1.397885896 1.270091113

GGGCTCAG 1.083550978 1.446989163 1.265270071

GGGCACTT 1.079810259 1.450613075 1.265211667

GGGCACCT 1.067498925 1.458938909 1.263218917

GGGTTCTA 1.10846698 1.416981517 1.262724249

GGGTTTGT 1.174150246 1.330408522 1.252279384

GGGGCACT 1.087181723 1.41628219 1.251731956

GGGCTTGA 1.108993039 1.393978237 1.251485638

GGGTCTGT 1.168399232 1.330529981 1.249464607

GGGGCCAG 1.079035261 1.416339438 1.24768735 GGGCTGTA 1.073693277 1.411997852 1.242845564

GGGTTGTC 1.180672138 1.303177442 1.24192479

GGGTCGAT 1.136383562 1.346527432 1.241455497

GGGCACGA 1.073479606 1.405753862 1.239616734

GGGGTCTA 1.07770371 1.395716039 1.236709874

GGGGCAGG 1.119865233 1.351089406 1.23547732

GGGAAGGG 1.094235901 1.374881299 1.2345586

GGGCTCAT 1.034702298 1.433421224 1.234061761

GGGGAGAG 1.144693885 1.32310795 1.233900917

GGGTGCTC 1.096554411 1.369337594 1.232946002

GGGTCTGC 1.120569558 1.341568569 1.231069064

GGGGTTTC 1.059567724 1.399014889 1.229291306

GGGGCACA 1.07537414 1.382779119 1.22907663

GGGTCCAC 1.087165713 1.368099563 1.227632638

GGGCGGAT 1.143897601 1.302920384 1.223408993

GGGCTTCA 1.048653706 1.396143062 1.222398384

GGGTTGCT 1.097503169 1.346564738 1.222033954

GGGCGTCA 1.057975588 1.385956928 1.221966258

GGGCGGAG 1.139597345 1.303579098 1.221588222

GGGGAGCG 1.113751009 1.321392676 1.217571842

GGGCCTAA 1.081511546 1.35186859 1.216690068

GGGTTGCA 1.102717756 1.330425901 1.216571829

GGGCTGTG 1.026581915 1.404032314 1.215307115

GGGTGGGA 1.049911965 1.37551789 1.212714927

GGGACCGA 0.997250078 1.424363886 1.210806982

GGGCTTTG 1.034582443 1.386361267 1.210471855

GGGACGTC 1.092347032 1.32778294 1.210064986

GGGCTTTC 1.020506625 1.396714087 1.208610356

GGGACGCC 1.067032646 1.34947504 1.208253843 GGGGTCAC 1.025730565 1.389775354 1.207752959

GGGCGTGG 1.074736411 1.340669667 1.207703039

GGGTCCTA 1.056156973 1.355138292 1.205647633

GGGGCTTA 1.047722617 1.35989151 1.203807063

GGGTCGTG 1.144914247 1.26241838 1.203666314

GGGTGCAC 1.073059152 1.327651836 1.200355494

GGGCCTAG 1.049551502 1.350267437 1.19990947

GGGGCTAA 1.060830325 1.338016321 1.199423323

GGGGTCTG 1.07949421 1.31230278 1.195898495

GGGCGTCT 1.045495282 1.343550809 1.194523046

GGGAGGCC 1.083471734 1.303907519 1.193689627

GGGCGTTA 1.041864756 1.342913734 1.192389245

GGGAGCGT 1.024756678 1.358610631 1.191683654

GGGCTCAC 0.973767502 1.407525024 1.190646263

GGGAGCGA 1.048623591 1.331447921 1.190035756

GGGCTTCT 1.002998502 1.37609706 1.189547781

GGGTCTGG 1.120082901 1.258800862 1.189441881

GGGTCGTT 1.076164666 1.301911661 1.189038163

GGGTGCGA 1.055794911 1.315381323 1.185588117

GGGGAGCA 1.059627868 1.310097534 1.184862701

GGGGCACC 1.041077031 1.327330484 1.184203757

GGGTTCTT 1.03655947 1.329219528 1.182889499

GGGTCTTT 1.075779556 1.289333911 1.182556734

GGGAGCTC 1.051807625 1.310936225 1.181371925

GGGACCCT 0.984989653 1.37563794 1.180313797

GGGGCCAC 1.014423285 1.345965945 1.180194615

GGGACCCA 1.018652415 1.336316472 1.177484443

GGGTCTTC 1.057862556 1.293579131 1.175720844

GGGCTTCC 0.985771983 1.364993035 1.175382509 GGGCATCA 1.021133791 1.320958336 1.171046064

GGGTGCGT 1.015748695 1.321670256 1.168709476

GGGTTCAC 1.012140577 1.323796196 1.167968386

GGGCGGAA 1.073479606 1.25990523 1.166692418

GGGTTTTG 1.079161164 1.251012031 1.165086597

GGGCACCC 0.996907893 1.332285715 1.164596804

GGGGCCAT 1.018404942 1.309642292 1.164023617

GGGTCCAG 1.033840886 1.293167351 1.163504118

GGGCGTGT 0.998511908 1.327167071 1.16283949

GGGCACAA 0.95477195 1.369897556 1.162334753

GGGTCCTT 0.989122431 1.333926058 1.161524245

GGGCCTCA 1.00267392 1.318974929 1.160824425

GGGTTTTC 1.048451374 1.269810762 1.159131068

GGGGTGGT 1.027405951 1.290688353 1.159047152

GGGTCCAT 1.026196378 1.291101565 1.158648971

GGGAGCGG 1.045556118 1.269390542 1.15747333

GGGCTGCG 1.000984121 1.308322475 1.154653298

GGGTTCAG 1.051412382 1.253641003 1.152526693

GGGCTTGT 1.005842908 1.298330557 1.152086733

GGGACCGC 0.973714398 1.329109838 1.151412118

GGGTCCTG 1.026992875 1.2754272 1.151210037

GGGTTCTG 1.051252644 1.248374356 1.1498135

GGGGAGGT 1.021254108 1.277869939 1.149562024

GGGGCTCA 0.962698256 1.335413474 1.149055865

GGGGAGCT 1.020731817 1.275151571 1.147941694

GGGCTGTC 1.00642428 1.289134608 1.147779444

GGGTTGCG 1.06291599 1.23197112 1.147443555

GGGCGTGA 1.010358685 1.279817359 1.145088022

GGGTTTGG 1.048680563 1.223148221 1.135914392 GGGTCGTA 1.05954625 1.209845371 1.13469581

GGGTGCGC 0.99111156 1.277023002 1.134067281

GGGGACCG 0.999998237 1.265711609 1.132854923

GGGCTCCT 0.916274365 1.339722999 1.127998682

GGGTTCTC 0.989219527 1.262031095 1.125625311

GGGACCCG 0.937824993 1.304637687 1.12123134

GGGTCTCC 0.980603234 1.258212296 1.119407765

GGGGTGGA 1.02173738 1.211699253 1.116718317

GGGACGGT 1.009140624 1.2237037 1.116422162

GGGAGGGA 1.00545809 1.225897346 1.115677718

GGGCTTGG 0.978315755 1.252961492 1.115638623

GGGGCTCG 0.960048811 1.267765298 1.113907054

GGGCTGCA 0.966499807 1.26081142 1.113655613

GGGGCTGA 0.998684764 1.228189136 1.11343695

GGGGTCGA 1.006955321 1.219416331 1.113185826

GGGTCGAC 1.033424019 1.189919462 1.111671741

GGGCGACC 0.853786195 1.368623048 1.111204621

GGGGTCTC 0.981640086 1.240419044 1.111029565

GGGCCTGT 0.954429934 1.264414682 1.109422308

GGGTTTCG 1.009372337 1.207087957 1.108230147

GGGCTGGT 0.908192758 1.306024375 1.107108566

GGGCCTGA 0.939169229 1.274630364 1.106899797

GGGGCCAA 0.927602584 1.279855841 1.103729212

GGGTAGGG 1.005037081 1.200303866 1.102670474

GGGCTCCA 0.914044685 1.290965711 1.102505198

GGGACCGT 0.909520933 1.293569461 1.101545197

GGGGTCCA 0.94841533 1.251581508 1.099998419

GGGTTTGC 0.988912996 1.199883096 1.094398046

GGGCGTTG 0.961520386 1.226942067 1.094231227 GGGAGGGC 0.961223645 1.223039702 1.092131673

GGGCTGCT 0.929174437 1.25308631 1.091130374

GGGGTGCA 0.977028531 1.204108783 1.090568657

GGGCCTCT 0.90115578 1.279930452 1.090543116

GGGCGGTA 0.976285204 1.204363584 1.090324394

GGGTCTTG 0.991685294 1.188074897 1.089880096

GGGCGTTC 0.921665244 1.255307591 1.088486418

GGGCAGGG 0.973087969 1.203814298 1.088451133

GGGTTCCT 0.942066454 1.231055394 1.086560924

GGGGTGCT 0.929735435 1.241252535 1.085493985

GGGAGCGC 0.952502286 1.216988355 1.08474532

GGGGTGCC 0.966734804 1.201995945 1.084365374

GGGACGCT 0.920780822 1.247139594 1.083960208

GGGGTCCT 0.939870134 1.22604622 1.082958177

GGGAGGGT 0.948808819 1.212253659 1.080531239

GGGCTGGA 0.938274017 1.219411352 1.078842685

GGGGTCGT 0.994642772 1.162530341 1.078586557

GGGGTCTT 0.957261882 1.198948142 1.078105012

GGGCGTGC 0.911203711 1.242424505 1.076814108

GGGCACCG 0.91268158 1.240659903 1.076670741

GGGTTGGG 1.006581541 1.146161026 1.076371284

GGGCTTGC 0.893593894 1.252811574 1.073202734

GGGTCGGA 0.992036794 1.15231386 1.072175327

GGGCCTTA 0.929372204 1.214796023 1.072084114

GGGCTCAA 0.890721719 1.252645678 1.071683699

GGGCTGGC 0.917715312 1.221486368 1.06960084

GGGCTCTC 0.890955583 1.245786912 1.068371247

GGGGTCCG 0.903490503 1.232301046 1.067895774

GGGACCGG 0.879186277 1.254042059 1.066614168 GGGCTTCG 0.899384061 1.23104636 1.06521521

GGGTGCTG 0.932364182 1.195516541 1.063940362

GGGGCTTG 0.927196435 1.19987893 1.063537682

GGGTTCCA 0.925036438 1.201617426 1.063326932

GGGTCCTC 0.943430534 1.179903183 1.061666858

GGGACGGC 0.967680165 1.153156194 1.06041818

GGGCGTCG 0.96776185 1.151397589 1.05957972

GGGCTCTA 0.856511175 1.259755315 1.058133245

GGGGCGTA 0.909890987 1.204600524 1.057245755

GGGGCCTA 0.883236207 1.230431793 1.056834

GGGGCATC 0.901896295 1.211448076 1.056672185

GGGGTGCG 0.937392479 1.175165173 1.056278826

GGGTCCAA 0.885408491 1.225663291 1.055535891

GGGCGTTT 0.888170221 1.219461681 1.053815951

GGGGCCTG 0.904191381 1.197457744 1.050824562

GGGCCTGC 0.908801199 1.192023715 1.050412457

GGGCTCCG 0.843257115 1.248492192 1.045874653

GGGGCTGC 0.918031764 1.166834155 1.042432959

GGGGCTTC 0.897768 1.177575311 1.037671655

GGGGCTCT 0.869879285 1.199159931 1.034519608

GGGAGGCA 0.935882731 1.127178942 1.031530837

GGGGCGGA 0.996270812 1.059781739 1.028026275

GGGGCGAA 0.930809491 1.123842327 1.027325909

GGGGCGAT 0.87994859 1.172738055 1.026343322

GGGGAGGA 0.938248389 1.111623357 1.024935873

GGGGCTGT 0.899097366 1.142268382 1.020682874

GGGCTGTT 0.85513976 1.177802981 1.016471371

GGGATCCC 0.87730801 1.154737689 1.016022849

GGGTTGCC 0.935102602 1.096036689 1.015569646 GGGCCTCG 0.891243443 1.136088456 1.01366595

GGGACGCG 0.8736865 1.150303895 1.011995197

GGGCTCTG 0.837248409 1.172423868 1.004836138

GGGCGGCG 0.916914745 1.090148245 1.003531495

GGGAGGCG 0.904298318 1.100757116 1.002527717

GGGGAGGC 0.878414131 1.117803739 0.998108935

GGGGCTTT 0.837534723 1.158435252 0.997984987

GGGCGGTT 0.84498216 1.146853883 0.995918021

GGGTTCGT 0.899055179 1.091107476 0.995081327

GGGCGTCC 0.895943475 1.092641594 0.994292534

GGGGTCGG 0.888789915 1.098890383 0.993840149

GGGCTCGT 0.830576081 1.154782872 0.992679477

GGGTCCGA 0.84912367 1.132678409 0.990901039

GGGGCGGC 0.933071809 1.044640789 0.988856299

GGGTGCGG 0.871788106 1.104163662 0.987975884

GGGCGGTC 0.889992037 1.084987969 0.987490003

GGGCTGCC 0.834749396 1.139175973 0.986962684

GGGGCGAC 0.803619654 1.159795424 0.981707539

GGGCTCGA 0.859417969 1.103936126 0.981677047

GGGTTTCC 0.868571712 1.094250825 0.981411269

GGGAGGCT 0.869291009 1.093223556 0.981257282

GGGCCTGG 0.833944817 1.123631074 0.978787946

GGGGCCTT 0.810708251 1.145407929 0.97805809

GGGCCTTC 0.819760472 1.136175094 0.977967783

GGGCGGCT 0.866116754 1.087168826 0.97664279

GGGCTCGC 0.82444142 1.124662983 0.974552202

GGGGTCGC 0.858992102 1.09006089 0.974526496

GGGTCGCA 0.854300029 1.090303219 0.972301624

GGGGCGAG 0.857516099 1.083835219 0.970675659 GGGTCCGC 0.814087643 1.126444215 0.970265929

GGGCTCTT 0.786728814 1.152315864 0.969522339

GGGTTCCG 0.816643367 1.120744406 0.968693886

GGGGCTCC 0.824124433 1.111387204 0.967755819

GGGCGGTG 0.837420533 1.09135739 0.964388962

GGGTCGGC 0.897282185 1.029080267 0.963181226

GGGCCGAG 0.798690296 1.126292192 0.962491244

GGGGCGTG 0.827684187 1.093130149 0.960407168

GGGGCTGG 0.816249928 1.092526673 0.9543883

GGGTCCCT 0.811605917 1.091811308 0.951708612

GGGACGCA 0.852630305 1.0501131 0.951371702

GGGTCGCG 0.858621566 1.040809215 0.94971539

GGGTTCGA 0.834159779 1.06447304 0.94931641

GGGCCTCC 0.811641989 1.086433025 0.949037507

GGGGCCGC 0.78584837 1.107992012 0.946920191

GGGGCCTC 0.774010918 1.113836447 0.943923683

GGGCTCGG 0.799434473 1.087959593 0.943697033

GGGTGCCC 0.845580808 1.037706156 0.941643482

GGGACGGG 0.817694638 1.06150595 0.939600294

GGGTCCCG 0.805077709 1.073884815 0.939481262

GGGTCCGT 0.810489574 1.061177877 0.935833726

GGGCCCAT 0.780212766 1.086413565 0.933313166

GGGCCTTT 0.77228643 1.086813883 0.929550156

GGGCCTTG 0.799233757 1.053826346 0.926530052

GGGCGGGT 0.779310194 1.071312563 0.925311378

GGGCCGGA 0.798490332 1.051993114 0.925241723

GGGCCGAT 0.760764872 1.082402123 0.921583497

GGGGCCGG 0.797219068 1.035586724 0.916402896

GGGCTGGG 0.780204752 1.043646649 0.911925701 GGGTCGGT 0.807361829 1.015103878 0.911232853

GGGCCGAC 0.752892612 1.067629274 0.910260943

GGGTCGTC 0.835149633 0.984849438 0.909999535

GGGCGGGC 0.763593625 1.049598695 0.90659616

GGGTCGCT 0.778604562 1.027981709 0.903293135

GGGGCGTT 0.766928813 1.03297024 0.899949526

GGGGTTTT 0.790317204 1.005793826 0.898055515

GGGCCCAG 0.74053741 1.053102626 0.896820018

GGGCGCAT 0.771005907 1.019213378 0.895109643

GGGGCGGT 0.833889892 0.949555275 0.891722584

GGGCCCAC 0.745432108 1.03150701 0.888469559

GGGCCGTA 0.760475971 1.013472627 0.886974299

GGGGCCGA 0.699252229 1.073905021 0.886578625

GGGTCCGG 0.761879742 1.00688192 0.884380831

GGGCCGAA 0.717074829 1.041923839 0.879499334

GGGCGGGA 0.793469355 0.960662694 0.877066024

GGGCGGCA 0.776347119 0.973290444 0.874818782

GGGTTCGC 0.757070562 0.984833364 0.870951963

GGGCCGTT 0.72548859 1.010081113 0.867784851

GGGCATCG 0.750197435 0.983874672 0.867036053

GGGTTCGG 0.764944353 0.949135631 0.857039992

GGGTACCC 0.735316161 0.975439679 0.85537792

GGGGCGTC 0.778874069 0.92958646 0.854230264

GGGCCCTC 0.682599222 1.025028238 0.85381373

GGGCCCTT 0.695481969 1.010491834 0.852986901

GGGGCGCA 0.721711781 0.976572059 0.84914192

GGGCGGCC 0.729520176 0.962493436 0.846006806

GGGCCGCT 0.688717973 0.998708921 0.843713447

GGGCGCAG 0.739575714 0.946966516 0.843271115 GGGCCGCA 0.693272675 0.990779033 0.842025854

GGGGACCC 0.725687048 0.956626381 0.841156714

GGGCTTTT 0.716126735 0.947968018 0.832047376

GGGTCGGG 0.748170268 0.900045686 0.824107977

GGGGCCCG 0.666577944 0.98116379 0.823870867

GGGGCCGT 0.696814402 0.925568305 0.811191354

GGGCCGCG 0.67504302 0.941603598 0.808323309

GGGCCCGC 0.643118441 0.958669926 0.800894184

GGGCCCTG 0.630391381 0.970728917 0.800560149

GGGCCCAA 0.670374828 0.928175342 0.799275085

GGGCCCTA 0.635446263 0.950086716 0.792766489

GGGCCGGT 0.648684661 0.912764914 0.780724788

GGGCGCAA 0.642797558 0.918165346 0.780481452

GGGCCCGT 0.620196502 0.931155704 0.775676103

GGGGCGCG 0.657008366 0.893797382 0.775402874

GGGCCGCC 0.663090757 0.878640912 0.770865835

GGGCCGGC 0.637532266 0.902223067 0.769877666

GGGGCCCA 0.645771951 0.890534695 0.768153323

GGGGCGCT 0.630096445 0.876843404 0.753469925

GGGCGCAC 0.628966893 0.848755356 0.738861125

GGGGTGGG 0.639463276 0.837036022 0.738249649

GGGCCGTG 0.617249474 0.854371572 0.735810523

GGGCCGGG 0.626902228 0.840367143 0.733634686

GGGTCCCA 0.654146184 0.808181444 0.731163814

GGGGCGGG 0.639794668 0.821578808 0.730686738

GGGCGCCT 0.593439868 0.865301703 0.729370785

GGGCCGTC 0.631923712 0.824867234 0.728395473

GGGCGCCA 0.606296915 0.849657615 0.727977265

GGGGCCCT 0.62498374 0.818075426 0.721529583 932 GGGGCGCC 0.633242621 0.808583459 0.72091304

933 GGGCCCGG 0.577208362 0.851008666 0.714108514

934 GGGCGCCG 0.582330054 0.820782469 0.701556262

935 GGGGAGGG 0.621337097 0.770629388 0.695983242

936 GGGCGCTA 0.581578527 0.798615425 0.690096976

937 GGGCGCTT 0.555824449 0.808696826 0.682260637

938 GGGTCGCC 0.611183748 0.753207192 0.68219547

939 GGGCGCGA 0.534214373 0.809461337 0.671837855

940 GGGCGCGT 0.573492984 0.754941686 0.664217335

941 GGGCTCCC 0.526359442 0.770270271 0.648314856

942 GGGCCCGA 0.514554248 0.78083549 0.647694869

943 GGGCGCGG 0.545982711 0.748550461 0.647266586

944 GGGCGCTG 0.525524266 0.737397388 0.631460827

945 GGGCGCTC 0.48781661 0.704183023 0.595999816

946 GGGCGCGC 0.463809507 0.697426999 0.580618253

947 GGGGTCCC 0.486124521 0.644497285 0.565310903

948 GGGCGCCC 0.407405312 0.619372279 0.513388795

949 GGGTTCCC 0.414025675 0.559109324 0.486567499

Example 2. Assessing the IVT activity of T7 promoter sequences

In vitro transcription assay (IVT)

One ng of dsDNA template (gBlocks Gene Fragments, Integrated DNA Technologies) was in vitro transcribed using the HiScribe T7 RNA Synthesis Kit (NEB E2040S, E2070S) producing 410 nucleotides long RNA (see SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8). RNA was purified with 1.6 volumes RNAClean XP beads, followed by elution in 20 pi water. RNA was quantified using the QubitTM RNA Assay Kit.

IVT-activitv of T7 promoter variants using a T7 RNA polymerase T7 promoter variants which ranked as comparatively high, moderate, or low self-transcribing in 5’RACE-Seq experiments as shown in Table 1; #4 (see SEQ ID NO:4), #368 (see SEQ ID NO: 6), #949 (see SEQ ID NO: 8), respectively, were exemplarily assayed for their activity in transcribing template DNA. The fold amplification of the template over time was higher, the higher the promoter ranked in 5’RACE-Seq (Figures 4 and 5). Assessment of the two other high and low ranked variants #1 (see SEQ ID NO:3) and #948 (see SEQ ID NO:7) also revealed high and low fold amplification of the template, respectively, as determined after 2h of IVT (Figure 6). This confirmed that the 5’RACE-Seq experiments were predictive of the efficiency/strength/activity of a given T7 promoter sequence in transcribing a downstream template sequence.

Furthermore, the transcripts increased rather linearly over time (Figure 5), demonstrating that the rank in 5’RACE-Seq experiments correlated well with the activity of the T7 promoter variant in linearly amplifying a template.

As shown exemplarily in Figure 7, the selection of a high-ranking downstream sequence (#4; see SEQ ID NO:4) revealed the improvement of the in vitro transcription activity of the T7 promoter over a low-ranking downstream sequence (#949; see SEQ ID NO: 8) at a range of IVT-template DNA concentrations as well as at saturating concentrations (40 ng). This suggested that selecting a suitable downstream flanking region can improve the performance of a T7 promoter also for large scale RNA synthesis.

The high-ranked promoter sequence #4 was further assayed with or without a 5 nucleotides long AT -rich upstream sequence (GAATT) after 2h of IVT. The directly adjacent upstream sequence in the controls was CAGGC (see SEQ ID NO:4, SEQ ID NO:8). It was found that replacing this control sequence by GAATT (see SEQ ID NO:5) further improved the activity of the T7 promoter (Figure 8).

Example 3. T7 promoter variants improve CEL-seq2

In vitro transcription assay (IVT) using CEL-seq mimics

The pre-synthesized double-stranded DNA templates harboring a T7 promoter variant mimicked cDNAs which are generated during the CEL-seq or CEL-seq2 protocol after mRNA capture to monitor the specific effect of the T7 promoter independent of the mRNA capturing step during CEL-seq(2); see SEQ ID NO: 10 and SEQ ID NO: 11. One pg (0.05 pg/mΐ) of those double stranded CEL-seq cDNA mimics was in vitro transcribed using the Hi Scribe T7 RNA Synthesis Kit. RNA was purified with 1.6 volumes RNAClean XP beads and eluted in 10 pi water. RNA was quantified using a High Sensitivity RNA ScreenTape (Agilent 5067-5579) on an Agilent Tapestation. IVT activity of T7 promoter sequences in CEL-seq mimics

It was found that the directly adj acent upstream sequence GAATT enhanced the relative activity of the T7 promoter (total RNA yield) with the directly adjacent downstream sequence “GGGATAAT” similarly in CEL-seq cDNA mimics (SEQ ID NO: 10 and SEQ ID NO: 11) as in standard IVT templates (SEQ ID NO:4 and SEQ ID NO:5), see Figure 9 (left and middle panels).

It was furthermore shown that addition of the directly adjacent upstream sequence GAATT (see SEQ ID NO: 11) improved the RNA yield especially when the starting material was scarce. When the starting material was 1 ng (50 pg/mΐ), the increase in yield was about 1.1 -fold. When only 1 pg (0.05 pg/mΐ) IVT-template was used, the improvement was about 1.5-fold (Figure 9, middle and right panels).

It was further revealed that the T7 promoter variant #4 (see SEQ ID NO: 10; cf. Table 1), in particular with the directly adjacent upstream flanking sequence GAATT (see SEQ ID NO: 11) had a strongly improved activity compared to the T7 promoter used in the original CEL-seq2 protocol (see SEQ ID NO:9). (Figure 10).

This suggested that the combination of a suitable downstream (e.g. GGGATAAT) and upstream sequences (e.g. GAATT) such as in SEQ ID NO:5 and SEQ ID NO: 11 can improve the transcription efficiency especially in applications where only a very small amount of template DNA is available, for example single-cell analyses such as single-cell sequencing.

Amplification of mRNA in CEL-seq2 experiments

CEL-seq2 experiments were performed with single K562 cells according to Hashimshony et al., Genome Biol. 2016 Apr 28; 17:77 until the in vitro transcription step and the amplified RNA was measured. Specifically, the cDNA from 10 cells was pooled and in vitro transcribed for 15h after reverse transcription and second strand cDNA synthesis. Different reverse transcription (RT) primers comprising T7 promoter variants were compared to the original CEL-seq2 RT primer (SEQ ID NO: 12). RT primers with T7 promoter variants ranking #4 and #66 as shown in Table 1 and further comprising “GAATT” as upstream sequence (SEQ ID NO: 13 and SEQ ID NO: 15, respectively) showed stronger activity than the original CEL-seq2 RT primer (SEQ ID NO: 12) (Figures 11 and 12). The #66 T7 promoter showed the strongest activity in this experiment (SEQ ID NO: 15), probably because it was the shortest primer as only two nucleotides had to be inserted (corresponding to positions +4 and +5) between the “GGG” at positions +1 to +3 and the directly adjacent sequencing adapter of the original CEL-seq2 primer (starting at position +6 and encompassing positions +6 to +8). Hence, reducing the length of RT primers may have a positive effect on mRNA capture and thus on the performance of CEL-seq(2) based methods.

This suggested that increasing the overlap of the +1 to +8 directly adjacent downstream flanking region of the T7 core promoter with the sequencing adapter may further improve the performance of CEL-seq or CEL-seq2 methods.

RT primers comprising #4 and #66 ranking sequences with “AATTG” as directly adjacent upstream sequence (SEQ ID NO: 14 and SEQ ID NO: 16, respectively) showed no improvement over the original CEL-seq2 RT primer having “GCCGG” as upstream sequence (SEQ ID NO: 12) (Figure 11)

This indicated that “GAATT” is advantageous, in particular in CEL-seq or CEL-seq2 experiments as upstream sequence directly adjacent to the T7 promoter as it had a positive effect on the strength of the T7 promoter.

CEL-Seq2 based single-cell RNA-sequencing

The full CEL-Seq2 protocol was performed as described in Hashimshony et ah, Genome Biol. 2016 Apr 28;17:77, and as illustrated in Figure 13, unless indicated differently. In particular, a modified reverse transcription primer (CEL-Seq+; SEQ ID NO: 15) was employed instead of the original (conventional) CEL-Seq2 reverse transcription primer (SEQ ID NO: 12) where indicated.

In brief, single cells were FACS sorted using a BD Aria III device into 96 well plates with barcoded reverse transcription primers (Figure 13) comprising either the modified T7 promoter (SEQ ID NO: 15) or the conventional CEL-Seq2 T7 promoter (0.4 mM) in lysis buffer (110 pM dNTPs, 0.007 % Triton X-100, 1.4 U SUPERaseln) and stored at -80°C. After thawing, plates were heated to 72°C for 3 min and incubated for 10 min at 10°C. Four pi Superscript II reaction mix were added at room temperature and the plate was incubated for 1 h at 42 °C followed by heat inactivation for 10 min at 70°C. Second strand synthesis was performed at 16 °C for 2 h with the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB E6111S). Single cell reactions were pooled separately for CEL-Seq2 or CEL-Seq+ primers, purified with 1.2 volumes of Ampure XP, and in vitro transcribed for 15 h using the MEGAscript T7 kit. After EXOSAP -IT treatment (Thermo, 78201) and fragmentation for 1.5min (NEB E6150S), RNA was cleaned up by 1.8 volumes of Ampure XP. The fragmented RNA was combined with HEX-primer and dNTPs and incubated at 65 °C for 5 min followed by addition of superscript II mix and incubation at 25°C for 10 min and 42 °C for 1 h. The cDNA was amplified with 12 cycles of PCR using KAPA HiFi HotStart ReadyMix. Final reactions were cleaned up with 0.8 fold Ampure XP and sequenced on a MiniSeq (Illumina) instrument.

Single-cell RNA-sequencing data analysis

Fastq files from each dataset were processed by zUMI software (version 2.5) for filtering, single cell demultiplexing and mapping the reads into genes. Filtering was done by using default parameters of zUMI (minimum reads per cell equal to 100), reads were mapped to hg38 genome assembly version and gencode (v27) annotation for gene read count. From each dataset, the output matrix files from zUMI were accessed by Seurat (version 3). Reads from intronic and exonic region of the genes were included and no further coverage filtering was applied.

The CEL-Seq+ reverse transcription primer (SEP ID NO: 15) improves sensitivity and accuracy of CEL-Seq2 based single-cell RNA sequencing

Successful preparation of a sequencing library strongly depends on the efficiency of the T7- based amplification step. A main characteristic of conventional single-cell RNA-seq (scRNA- seq) is a high “drop out” rate, which means that features (e.g. transcripts) that are present in an individual cell escape detection. Accordingly, conventional scRNA-seq data is inherently shallow, meaning that most expressed genes are only represented by a small number of transcript counts, and some are not represented by any transcript count at all (missed expressed genes). This is because many mRNA molecules are not initially captured by hybridization or are subsequently lost in one of the downstream steps of library preparation, all of which are not 100% efficient.

It was thus tested whether additional copies generated by a more efficient T7 promoter (e.g. SEQ ID NO:20) may increase the probability especially of lowly abundant transcripts to be represented in the final sequencing output, corresponding to enhanced sensitivity of scRNA- seq. It was found that the CEL-Seq+ reverse transcription primer (SEQ ID NO: 15) harboring a modified and more active T7 promoter (SEQ ID NO:20) facilitated library preparation, and substantially increased library complexity and the number of expressed genes detected per cell, highlighting a particular value for bioanalytical applications (Figures 11 to 17), as detailed out in the following.

Following mRNA capture with the modified reverse transcription primer (SEQ ID NO: 15), a robust ~2.5-fold increase in aRNA amplification from pooled cDNA of 10 single K562 cells was observed, which facilitated library preparation from scarce material (Figures 12 and 13). Moreover, employing the modified reverse transcription primer (SEQ ID NO: 15) harboring the modified T7 promoter sequence (SEQ ID NO:20) for CEL-seq2 based scRNA-seq (CEL-seq+) substantially increased the number of detected genes (average 9749 vs. 8281), and the number of unique molecular identifiers (UMIs; average 85066 vs. 53541) from single cells compared to the original CEL-seq2 reverse transcription primer (SEQ ID NO: 12) (Figure 14). Detection of almost 10,000 genes per cell likely approaches the entire set of expressed genes present in a single cell at any given time. In addition to higher detection rates, expression levels of individual genes were measured with higher accuracy by CEL-Seq+ across the entire gene expression spectrum, as reflected by higher UMI counts and a consistently lower coefficient of variation (CV) (Figures 14 and 15). As an additional benchmark the genes detected in deep sequenced bulk K562 RNA-Seq (Prost (2015), Nature 525) were divided into expression quartiles and their recovery in CEL-seq2 vs. CEL-Seq+ was compared. Enhanced recovery of genes, in particular from lower expression quartiles, in CEL-Seq+ suggested that improved linear amplification of RNA molecules based on an improved T7 promoter sequence (e.g. SEQ ID NO:20 or SEQ ID NO: 18) directly translates into substantially increased sensitivity of single-cell genomics applications (Figure 16). At the same time, the detection rate of transcription factors, chromatin binders, or genes involved in Chronic myelogenous leukemia (CML) disease progression was significantly increased (Figure 17). Specifically, 64 transcription factors were exclusively detected by CEL-Seq2+, including many with known relevance for leukemia (i.e. TLX21), CML tumor stem cells (i.e. PPARG20), CML cancer cell metabolism (i.e. SIX120) or response to treatment (i.e. PBX120). Accordingly, drug development and diagnostics, e.g. in the field of tumor biology, may broadly benefit from application of an improved T7 promoter for scRNA-seq, i.e. such as in CEL-Seq+.

Further single-cell DNA- and RNA-sequencing approaches and nucleic acids based diagnostics of scarce clinical material

Furthermore, since important low-abundant regulatory genes are usually missed by microdroplet-based single-cell RNA-sequencing methods that produce particularly shallow transcriptomes, microdroplet-based methods such as inDrop (Klein, A. M. etal. , Cell 161,1187- 1201, 2015) may benefit from employing the herein described improved T7 promoters (e.g. SEQ ID NO:20 or SEQ ID NO: 18) to increase gene detection rates.

In general, since uniform and accurate amplification of nucleic acids is important when starting material is scarce and precious, the improved T7 promoter sequences provided herein (e.g. SEQ ID NO:20 or SEQ ID NO: 18) can be readily applied in various IVT-based methods to boost linear amplification of nucleic acids. This is of particular interest for various single-cell DNA- and RNA-sequencing approaches, and for nucleic acids based diagnostics of scarce clinical material such as circulating tumor cells (CTCs) (Lawson et al., Nat. Cell Biol. 20,1349- 1360 (2018)).

Claims

1. A method for in vitro transcribing (IVT) RNA, comprising

(a) providing a DNA polynucleotide as an IVT template comprising (i) a T7 promoter sequence comprising

(1) a T7 core promoter sequence and

(2) a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising three guanines at positions +1 to +3, an adenine or thymine at position +4, at least two adenines at positions +4 to +8 and/or a thymine at position +4, and at most one cytosine at positions +4 to +8, and downstream thereof (ii) a nucleotide sequence encoding the RNA to be transcribed, and

(b) in vitro transcribing said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.

2. The method of claim 1, wherein the IVT template is present at a concentration of less than 1 ng/mΐ, preferably of less than 100 pg/mΐ, preferably of less than 10 pg/pl.

3. The method of claims 1 or 2, wherein step (a) comprises reverse transcribing an RNA to cDNA, wherein said cDNA is the IVT template, and wherein a DNA polynucleotide comprising said T7 promoter sequence is used as reverse transcription primer, and wherein step (b) comprises transcribing said cDNA to RNA.

4. The method of claim 3, wherein the reverse transcription primer comprises a poly-T- stretch, wherein the RNA that is reverse transcribed to cDNA is an mRNA, the in vitro transcribed RNA is an antisense RNA (aRNA), and wherein the polyT-stretch is a nucleotide sequence of 9 to 35 consecutive thymines.

5. A method for amplifying a target polynucleotide, wherein said method comprises the steps of

(a) synthesizing the complementary strand of a target polynucleotide, wherein said synthesizing comprises annealing a DNA polynucleotide comprising the T7 promoter sequence according to any of claims 1 to 4 to said target polynucleotide, thereby obtaining an IVT template comprising said T7 promoter and a nucleotide sequence downstream thereof, i.e. wherein said nucleotide sequence reflects the target polynucleotide sequence, and

(b) in vitro transcribing said IVT template according to any of claims 1 to 4, thereby amplifying said target polynucleotide.

6. The method of claim 3, wherein the target polynucleotide is an RNA which is reverse- transcribed into cDNA, thereby amplifying said RNA as aRNA.

7. The method of any of the preceding claims, wherein the IVT template is obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from less than 100 cells, preferably less than 10 cells.

8. The method of any of the preceding claims, wherein the IVT template is obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from a single cell.

9. The method of any of the preceding claims, wherein the IVT template is obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from a liquid biopsy and/or a body fluid.

10. The method of any of the preceding claims, wherein the IVT template is obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide, wherein said RNA and/or at least one target polynucleotide is present at a concentration of less than 1 ng/mΐ, preferably of less than 100 pg/pl, preferably of less than 10 pg/pl.

11. A method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene comprising the steps of

(a) reverse transcribing an RNA to the first strand of a cDNA comprising annealing a DNA polynucleotide comprising the T7 promoter according to any one of the preceding claims; and

(b) transcribing the cDNA into aRNA by using a T7 polymerase.

12. The method of claim 11, further comprising a step (c) of generating a double-stranded cDNA from the aRNA of step (b), and/or a step of sequencing the aRNA of step (b) and/or the cDNA of step (c).

13. A method for determining the partial or full nucleotide sequence(s) and/or abundance of at least one target polynucleotide comprising the steps of

(a) amplifying at least one target polynucleotide according to any of claims 5 to 10, and

(b) determining the partial or full nucleotide sequence(s) and/or abundance of said at least one amplified target polynucleotide.

14. A method for determining the transcript level of at least one gene comprising the steps of

(a) amplifying at least one target polynucleotide according to any of claims 5 to 10, wherein said target polynucleotide is an mRNA corresponding to said at least one gene, and

15. The method of any of claims 11 to 14, wherein the partial or full nucleotide sequence(s), the abundance of at least one target polynucleotide and/or the transcript level of at least one gene is determined in a sample comprising less than 100 cells, preferably less than 10 cells, preferably a single cell.

16. The method of any of claim 11 to 15, for determining the partial or full nucleotide sequence(s) of an RNA contained in a single cell or single cell equivalent and/or the transcript level of at least one gene of a single cell or single cell equivalent.

17. The method of any of claims 11 to 16, wherein the partial or full nucleotide sequence(s), the abundance of at least one target polynucleotide and/or the transcript level of at least one gene is determined in a sample, wherein said mRNA and/or at least one target polynucleotide is present at a concentration of less than 1 ng/mΐ, preferably of less than 100 pg/mΐ, preferably of less than 10 pg/pl.

18. A DNA polynucleotide, comprising a T7 promoter sequence comprising

(1) a T7 core promoter sequence and

(2) a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising three guanines at positions +1 to +3, an adenine or thymine at position +4, at least two adenines at positions +4 to +8 and/or a thymine at position +4, and at most one cytosine at positions +4 to +8.

19. The method of any of claims 1 to 17, or the DNA polynucleotide of claim 18, wherein the downstream flanking region further comprises at least three adenines or thymines at positions +4 to +8 and/or a guanine followed by an adenine within positions +5 and +6, and the downstream flanking region does not comprise three consecutive thymines within positions +4 to +8, and/or two consecutive thymines within positions +4 and +7, when a cytosine or guanine is present at positions +4 to +8, or a thymine, cytosine or guanine is present at position +4; a thymine followed by a cytosine within positions +4 and +7; a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8; and a cytosine followed by a guanine within positions +4 to +7.

20. The method or the DNA polynucleotide of claim 19, wherein the downstream flanking region further comprises an adenine at position +4 and at most one guanine or cytosine at positions +4 to +8.

21. The method or the DNA polynucleotide of claim 20, wherein the downstream flanking region does not comprise a thymine at position +5 followed by a guanine at position +6 and two consecutive thymines within positions +4 to +8.

22. The method or the DNA polynucleotide of claim 21, wherein the downstream flanking region further comprises an adenine at position +4, three to four adenines at positions +4 to +8, and no guanines or cytosines at positions +4 to +8, preferably wherein the downstream flanking region has the sequence GGGATAAT.

23. The method of any of claims 1 to 20 or the DNA polynucleotide of any of claims 22 to 24, wherein the downstream flanking region further comprises the sequence AGT at positions +6 to +8.

24. The method or the DNA polynucleotide of claim 23, wherein the downstream flanking region has the sequence GGGAAAGT, GGGTAAGT, GGGATAGT, GGGTGAGT, or GGGAGAGT, preferably GGGAGAGT.

25. The method of any of claims 1 to 19 or the DNA polynucleotide of claims 18 or 19, wherein the downstream flanking region further comprises the sequence AG at positions +7 to +8.

26. The method or the DNA polynucleotide of claim 25, wherein the downstream flanking region has the sequence GGGAAAAG, GGGAATAG, GGGATAAG, GGGTGAAG, GGGTAAAG, GGGAGAAG, GGGTGTAG, GGGAGTAG, or GGGATTAG.

27. The method of any of claims 1 to 17 or 19 to 26 or the DNA polynucleotide of any of claims 18 to 26, wherein the T7 promoter further comprises the sequence, AATT, preferably GAATT, directly upstream of the T7 core promoter sequence.

28. The method of any of claims 1 to 17 or 19 to 27 or the DNA polynucleotide of any of claims 18 to 27, wherein the DNA polynucleotide is a reverse transcription primer, further comprising a polyT-stretch, wherein the polyT-stretch is a nucleotide sequence of 9 to 35 consecutive thymines, preferably 22 to 27 consecutive thymines, preferably 24 consecutive thymines, followed by an adenine, cytosine or guanine.

29. The method of any of claims 1 to 17 or 19 to 28 or the DNA polynucleotide of any of claims 18 to 28, wherein the DNA polynucleotide is a reverse transcription primer, further comprising a sequencing adapter, wherein the sequencing adapter is a nucleotide sequence comprising a nucleotide sequence comprised in a library preparation primer, wherein the library preparation primer comprises a flow cell binding sequence, which is complementary to an oligo- or polynucleotide present at the surface of the flow cell of a next-generation-sequencing device, and wherein the sequencing adapter does not comprise more than eight consecutive thymines and/or only thymines.

30. The method or the DNA polynucleotide of claim 29, wherein the sequencing adapter is downstream of the T7 core promoter, and wherein the polyT-stretch is downstream of the sequencing adapter and not overlapping with the sequencing adapter.

31. The method of any of claims 5 to 17, or 19 to 30, wherein at least one target polynucleotide is amplified from less than 100 cells, preferably less than 10 cells, preferably a single cell, and wherein the T7 promoter further comprises the sequence AATT directly upstream of the T7 core promoter sequence.

32. The method of claim 31, wherein an mRNA is amplified, and wherein the T7 promoter further comprises a poly-T-stretch according to claims 28 or 30.

33. The method of any of claims 5 to 17 or 19 to 32, wherein the target polynucleotide is from a prokaryote or a eukaryote, preferably an animal or a human.

34. The method of any of claims 1 to 17, or 19 to 33 or the DNA polynucleotide of any of claims 18 to 30, wherein the DNA polynucleotide further comprises downstream of the T7 core promoter sequence a nucleotide sequence encoding an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPR

RNA.

35. A kit comprising the DNA polynucleotide of any one of claims 18 to 30, further comprising a T7 polymerase, one or more modified and/or unmodified ribonucleotide triphosphate(s), library preparation primer(s), sequencing primer(s), and/or a microfluidic chip.