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WO2025062001A1 - Séquençage optimisé d'acides nucléiques - Google Patents

Séquençage optimisé d'acides nucléiques Download PDF

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Publication number
WO2025062001A1
WO2025062001A1 PCT/EP2024/076524 EP2024076524W WO2025062001A1 WO 2025062001 A1 WO2025062001 A1 WO 2025062001A1 EP 2024076524 W EP2024076524 W EP 2024076524W WO 2025062001 A1 WO2025062001 A1 WO 2025062001A1
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WIPO (PCT)
Prior art keywords
immobilised
sequencing
primer
solid support
primers
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PCT/EP2024/076524
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English (en)
Inventor
Aathavan KARUNAKARAN
Seth MCDONALD
Merek SIU
Nileshi SARAF
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Illumina, Inc.
Marks & Clerk Llp
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Application filed by Illumina, Inc., Marks & Clerk Llp filed Critical Illumina, Inc.
Publication of WO2025062001A1 publication Critical patent/WO2025062001A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • aspects relate to solid supports and methods for use in nucleic acid sequencing, in particular solid supports and methods for use in concurrent sequencing.
  • the invention also relates to methods and kits for use in nucleic acid sequencing, in particular methods for use in optimising the signal-to-noise ratio in simultaneous sequencing, in particular by using a calculated amount of terminated primer(s).
  • deoxyribonucleic acid analogs conjugated to fluorescent labels are hybridized to the template nucleic acids, and excitation light sources are used to excite the fluorescent labels on the deoxyribonucleic acid analogs.
  • Detectors capture fluorescent emissions from the fluorescent labels and identify the deoxyribonucleic acid analogs.
  • the sequence of the template nucleic acids may be determined by repeatedly performing such sequencing cycles.
  • NGS allows for the sequencing of a number of different template nucleic acids simultaneously, which has significantly reduced the cost of sequencing in the last twenty years.
  • a solid support comprising: a plurality of first immobilised primers, wherein a proportion of the first immobilised primers are configured to be cleavable under first cleavage conditions; a plurality of second immobilised primers, wherein a proportion or substantially all of the second immobilised primers are configured to be cleavable under second cleavage conditions; and wherein the proportion of the first immobilised primers configured to be cleavable under first cleavage conditions is less than the proportion of the second immobilised primers configured to be cleavable under second cleavage conditions.
  • the solid support comprises at least one well, and wherein the plurality of first immobilised primers and the plurality of second immobilised primers are located within the well. In one aspect, the solid support comprises a plurality of wells, wherein the plurality of first immobilised primers and the plurality of second immobilised primers are located within each of the wells.
  • the proportion of first immobilised primers are configured to be cleavable by a thermal trigger, a light trigger, and/or a chemical/biochemical trigger. In one aspect, the proportion of first immobilised primers are configured to be cleavable by a glycosylase. In one aspect, the proportion of first immobilised primers are configured to be cleavable by a uracil glycosylase or an oxoguanine glycosylase (e.g. 8-oxoguanine glycosylase). In one aspect, the proportion of first immobilised primers are configured to be cleavable by an oxoguanine glycosylase (e.g. 8-oxoguanine glycosylase).
  • each first immobilised primer that is cleavable comprises a nucleobase which is not selected from guanine, cytosine, adenine or thymine when the first immobilised primer is a DNA sequence; or wherein each first immobilised primer that is cleavable comprises a nucleobase which is not selected from guanine, cytosine, adenine or uracil when the first immobilised primer is an RNA sequence.
  • each first immobilised primer that is cleavable comprises oxoguanine (e.g. 8-oxoguanine) or uracil when the first immobilised primer is a DNA sequence, or wherein each first immobilised primer that is cleavable comprises oxoguanine (e.g. 8- oxoguanine) when the first immobilised primer is an RNA sequence.
  • each first immobilised primer that is cleavable comprises oxoguanine (e.g. 8-oxoguanine) when the first immobilised primer is a DNA sequence.
  • the proportion of second immobilised primers are configured to be cleavable by a thermal trigger, a light trigger, or a chemical/biochemical trigger. In one aspect, the proportion of second immobilised primers are configured to be cleavable by a glycosylase. In one aspect, the proportion of second immobilised primers are configured to be cleavable by a uracil glycosylase or an oxoguanine glycosylase (e.g. 8- oxoguanine glycosylase). In one aspect, the proportion of second immobilised primers are configured to be cleavable by a uracil glycosylase.
  • each second immobilised primer that is cleavable comprises a nucleobase which is not selected from guanine, cytosine, adenine or thymine when the second immobilised primer is a DNA sequence; or wherein each second immobilised primer that is cleavable comprises a nucleobase which is not selected from guanine, cytosine, adenine or uracil when the second immobilised primer is an RNA sequence.
  • each second immobilised primer that is cleavable comprises oxoguanine (e.g. 8-oxoguanine) or uracil when the second immobilised primer is a DNA sequence, or wherein each second immobilised primer that is cleavable comprises oxoguanine (e.g. 8-oxoguanine) when the second immobilised primer is an RNA sequence
  • each first immobilised primer comprises a sequence as defined in SEQ ID NO. 1 or 5, or a variant or fragment thereof; and each second immobilised primer comprises a sequence as defined in SEQ ID NO. 2, or a variant or fragment thereof; or wherein each first immobilised primer comprises a sequence as defined in SEQ ID NO. 2, or a variant or fragment thereof; and each second immobilised primer comprises a sequence as defined in SEQ ID NO. 1 or 5, or a variant or fragment thereof.
  • Each second polynucleotide sequence may comprise a first adaptor sequence, wherein the first adaptor sequence comprises a portion, which is substantially complementary to the first immobilised primer (or is substantially complementary to the first immobilised primer).
  • the first adaptor sequence may be at a 3’-end of the second polynucleotide sequence.
  • a solution comprising a polynucleotide library prepared by ligating adaptor sequences to double-stranded polynucleotide sequences as described above may be flown across a flowcell.
  • a particular polynucleotide strand from the polynucleotide library to be sequenced comprising, in a 5’ to 3’ direction, a second primer-binding complement sequence 302 (e.g. P7), a first terminal binding site complement 303’ (e.g. SBS12), a forward strand of the sequence 101 , a second terminal sequencing primer binding site 304 (e.g. SBS3’) and a first primer-binding sequence 30T (e.g. P5’), may anneal (via the first primerbinding sequence 301’) to the first immobilised primer 201 (e.g. P5 lawn primer) located within a particular well 203 ( Figure 7A).
  • a second primer-binding complement sequence 302 e.g. P7
  • a first terminal binding site complement 303’ e.g. SBS12
  • a forward strand of the sequence 101 e.g. SBS3’
  • a second terminal sequencing primer binding site 304 e.g. SBS
  • the polynucleotide library may comprise other polynucleotide strands with different forward strands of the sequence 101.
  • Such other polynucleotide strands may anneal to corresponding first immobilised primers 201 (e.g. P5 lawn primers) in different wells 203, thus enabling parallel processing of the various different strands within the polynucleotide library.
  • first immobilised primers 201 e.g. P5 lawn primers
  • a new polynucleotide strand may then be synthesised, extending from the first immobilised primer 201 (e.g. P5 lawn primer) in a direction away from the substrate 204.
  • this generates a template strand comprising, in a 5’ to 3’ direction, the first immobilised primer 201 (e.g. P5 lawn primer) which is attached to the solid support 200, a second terminal sequencing primer binding site complement 304’ (e.g. SBS3), a forward strand of the template 10T (which represents a type of “first portion”), a first terminal sequencing primer binding site 303 (which represents a type of “first sequencing primer binding site”) (e.g. SBS12’), and a second primer-binding sequence 302’ (e.g. P7’) ( Figure 7B).
  • Such a process may utilise an appropriate polymerase, such as a DNA or RNA polymerase.
  • the polynucleotide strand from the polynucleotide library may then be dehybridised and washed away, leaving a template strand attached to the first immobilised primer 201 (e.g. P5 lawn primer) ( Figure 7C).
  • first immobilised primer 201 e.g. P5 lawn primer
  • the second primer-binding sequence 302’ (e.g. P7’) on the template strand may then anneal to a second immobilised primer 202 (e.g. P7 lawn primer) located within the well 203. This forms a “bridge” or “sequence bridge” ( Figure 7D).
  • the strand attached to the second immobilised primer 202 may then be dehybridised from the strand attached to the first immobilised primer 201 (e.g. P5 lawn primer) ( Figure 7F).
  • a subsequent bridge amplification cycle can then lead to amplification of the strand attached to the first immobilised primer 201 (e.g. P5 lawn primer) and the strand attached to the second immobilised primer 202 (e.g. P7 lawn primer).
  • the second primer-binding sequence 302’ e.g. P7’
  • the first primer-binding sequence 30T e.g. P5’
  • the second immobilised primer 202 e.g. P7 lawn primer
  • further bridge amplification cycles may be conducted to increase the number of first polynucleotide sequences and second polynucleotide sequences within the well 203.
  • the “first portion” corresponds with the forward strand of the template 10T
  • the “second portion” corresponds with the forward complement strand of the template 101.
  • the template provides information (e.g. identification of the genetic sequence, identification of epigenetic modifications) on the original target polynucleotide sequence.
  • a sequencing process e.g. a sequencing-by-synthesis or sequencing-by-ligation process
  • identification is meant here obtaining genetic information from the polynucleotide strand or polynucleotide strands. This may include identification of the genetic sequence of the polynucleotide strand or polynucleotide strands (i.e. sequencing). Furthermore, this may instead, or additionally, include identification of mismatched base pairs. In addition, this may instead, or additionally, include identification of any epigenetic modifications, for example methylation. Accordingly, “identification” may mean identification of the genetic sequence of the polynucleotide strand or polynucleotide strands, mismatched base pairs, and/or identification of any epigenetic modifications.
  • sequencing may be carried out using any suitable "sequencing-by- synthesis" technique, wherein nucleotides are added successively in cycles to the free 3' hydroxyl group, resulting in synthesis of a polynucleotide chain in the 5' to 3' direction.
  • the nature of the nucleotide added may be determined after each addition.
  • One particular sequencing method relies on the use of modified nucleotides that can act as reversible chain terminators. Such reversible chain terminators comprise removable 3' blocking groups.
  • the modified nucleotides may carry a label to facilitate their detection.
  • a label may be configured to emit a signal, such as an electromagnetic signal, or a (visible) light signal.
  • the label is a fluorescent label (e.g. a dye).
  • a fluorescent label e.g. a dye
  • the label may be configured to emit an electromagnetic signal, or a (visible) light signal.
  • One method for detecting the fluorescently labelled nucleotides comprises using laser light of a wavelength specific for the labelled nucleotides, or the use of other suitable sources of illumination.
  • the fluorescence from the label on an incorporated nucleotide may be detected by a CCD camera or other suitable detection means. Suitable detection means are described in PCT/US2007/007991 , the contents of which are incorporated herein by reference in their entirety.
  • the detectable label need not be a fluorescent label. Any label can be used which allows the detection of the incorporation of the nucleotide into the DNA sequence.
  • Each cycle may involve simultaneous delivery of four different nucleotide types to the array of template molecules.
  • different nucleotide types can be added sequentially and an image of the array of template molecules can be obtained between each addition step.
  • each nucleotide type may have a (spectrally) distinct label.
  • four channels may be used to detect four nucleobases (also known as 4- channel chemistry) ( Figure 8 - left).
  • a first nucleotide type e.g. A
  • a second nucleotide type e.g. G
  • a second label e.g. configured to emit a second wavelength, such as blue light
  • a third nucleotide type e.g. T
  • a third label e.g.
  • one channel may be used to detect four nucleobases (also known as 1 -channel chemistry) ( Figure 8 - right).
  • a first nucleotide type e.g. A
  • a second nucleotide type e.g. G
  • a third nucleotide type e.g. T
  • a non-cleavable label e.g. configured to emit the wavelength, such as green light
  • a fourth nucleotide type e.g. C
  • a label-accepting site which does not include the label.
  • the sequencing process comprises a first sequencing read and second sequencing read.
  • the first sequencing read and the second sequencing read may be conducted concurrently. In other words, the first sequencing read and the second sequencing read may be conducted at the same time.
  • the first and second sequencing strands, and the clusters generated therefrom are spatially unresolved. Accordingly, the methods of the present invention allow the simultaneous sequencing of spatially unresolved clusters.
  • the methods of the invention provide a method of sequencing without the need for a paired-end turn and cluster re-synthesis. This in turn reduces the time taken to sequence a target polynucleotide, thus improving even further the efficiency of the sequencing protocol when using the method of the present invention.
  • a paired-end turn refers to the sequence of stages required to effectively invert the sequence for the second read in paired-end reading, after sequencing read 1 ( Figure 7). The paired-end turn may be facilitated by a cycle of bridge amplification and linearization.
  • the present invention also eliminates the need for cluster re-synthesis of the first or second polynucleotide sequences for read 2 of conventional SBS workflows ( Figure 7).
  • a method of preparing a first and second polynucleotide sequence for concurrent sequencing comprising:
  • the method of the invention may further comprise hybridising the first polynucleotide sequence to first immobilised primers on a solid support and hybridising the second polynucleotide sequence to second immobilised primers on a solid support; and synthesising a plurality of first and second polynucleotide sequences by conducting an amplification reaction to extend the first and second immobilised primers.
  • measurement of a first and second sequencing intensity is carried out sequentially.
  • the measurement of a first signal intensity generated from the first polynucleotide sequence is first, and the measurement of a second signal intensity generated from the second polynucleotide sequence is second - or vice versa.
  • the order of application is immaterial, so long as the first and second sequencing primers are applied separately and sequentially.
  • the method comprises applying (i.e. flowing across the surface of the solid support) a plurality of first (or second) sequencing primers (also known as a read 1 sequencing primer).
  • the first sequencing primers hybridise to the first sequencing primer binding site (e.g. first terminal sequencing primer binding site 303).
  • the method comprises applying a plurality of labelled first sequencing primers, and measuring the signal intensity generated from binding of the first sequencing primers to the first polynucleotide strand. This is the measurement of the first signal intensity described above.
  • the method comprises applying (i.e. flowing across the surface of the solid support) a plurality of first (or second) sequencing primers, where the sequencing primers are not labelled and conducting an extension reaction, as described above, to extend the first (or second) sequencing primer.
  • the sequencing primer may be extended by a plurality of labelled nucleotides, which are subsequently detected. In one embodiment, the sequencing primer is extended by just one labelled nucleotide. Alternatively, the sequencing primer is extended by 2, 3, 4, 5 or more labelled nucleotides.
  • the label comprises a cleavable covalent bond.
  • cleavable covalent bond refers to a covalent bond that can be cleaved, for example, under the application of heat, light or other (bio)chemical methods (e.g. by exposure to a degradation agent, such as an enzyme or a catalyst), while a “non- cleavable covalent bond” is stable to degradation under such conditions.
  • cleavable covalent bonds include thermally or photolytically cleavable cycloadducts (e.g.
  • X is the desired ratio of signal intensities. In one embodiment X may be 2. tR1 P is terminated R1 primer
  • R1 p is Read 1 sequencing primer (non-terminated)
  • R1 si is a first signal intensity generated from a first nucleotide sequence
  • R2si is a second signal intensity generated from a first nucleotide sequence
  • first and second polynucleotide strands can be sequenced concurrently.
  • first sequence e.g. forward strand of the template
  • second sequence e.g. reverse strand of the template
  • sequencing errors are improved because concurrently received signals can be better distinguished by 16 QaM analysis. This is illustrated in Figures 11 B and 12.
  • improving spatially unresolved imaging for clusters by improving the SNR allows for smaller pitch and nanowell dimensions to be used.
  • improved SNR allows imaging technologies with lower numerical aperture to be used, allowing for faster imaging of larger areas. This in turn increases the throughput power of sequencing reads.
  • sequences to be identified comprise one or more index sequence
  • the index is typically read separately from read 1 and read 2. Either before read 1 and before read 2, or afterwards. If afterwards, the extended sequencing primer is denatured and washed off the flowcell, and index primer is hybridized for the several cycles of index read.
  • the first portions and second portions may be different polynucleotide sequences. That is, the sequences may be genetically unrelated and/or derived from different sources.
  • the single (concatenated) polynucleotide strand with a first and second portion may comprise a first sequencing primer binding site and a second sequencing primer binding site, (used to sequence the first and second portions respectively) where the first sequencing primer binding site and second sequencing primer binding site are of a different sequence to each other and bind different sequencing primers.
  • a method of preparing a polynucleotide sequence comprising a first portion and a second portion for concurrent sequencing, wherein the method comprises: measuring a first signal intensity generated from a first portion of the polynucleotide sequence, and measuring a second signal intensity generated from a second portion of the polynucleotide sequence, and based on the first signal intensity and the second signal intensity, a subsequent signal intensity generated from sequencing the first nucleotide strand can be or is attenuated, wherein the attenuated subsequent signal intensity generated from sequencing the first polynucleotide strand has a lower intensity than a subsequent signal intensity generated from sequencing the second polynucleotide strand.
  • the measured intensity of a first and second signal as described above can be used to calculate the amount of terminated first (or second) sequencing primer to be added.
  • the method comprises applying a calculated amount of first (or second) terminated primer. Any amount of terminated sequencing primer that modulates the signal intensity ratio towards 2:1 or around 2:1 can be used.
  • Figure 9 is a scatter plot showing an example of sixteen distributions of signals generated by polynucleotide sequences disclosed herein. By creating different intensities of signals between the two clusters their identities can be resolved, despite them being spatially unresolved, i.e. within the same CMOS pixel.
  • the scatter plot of Figure 9 shows sixteen distributions (or bins) of intensity values from the combination of a brighter signal (i.e. a first signal as described herein) and a dimmer signal (i.e. a second signal as described herein); the two signals may be co-localized and may not be optically resolved as described above.
  • the intensity values shown in Figure 9 may be up to a scale or normalisation factor; the units of the intensity values may be arbitrary or relative (i.e., representing the ratio of the actual intensity to a reference intensity).
  • the sum of the brighter signal generated by the first portions and the dimmer signal generated by the second portions results in a combined signal.
  • the combined signal may be captured by a first optical channel and a second optical channel. Since the brighter signal may be A, T, C or G, and the dimmer signal may be A, T, C or G, there are sixteen possibilities for the combined signal, corresponding to sixteen distinguishable patterns when optically captured. That is, each of the sixteen possibilities corresponds to a bin shown in Figure 9.
  • the computer system can map the combined signal generated into one of the sixteen bins, and thus determine the added nucleobase at the first portion and the added nucleobase at the second portion, respectively.
  • the computer processor base calls both the added nucleobase at the first portion and the added nucleobase at the second portion as C.
  • the processor base calls the added nucleobase at the first portion as C and the added nucleobase at the second portion as T.
  • the processor base calls the added nucleobase at the first portion as C and the added nucleobase at the second portion as G.
  • the processor base calls the added nucleobase at the first portion as C and the added nucleobase at the second portion as A.
  • the processor base calls the added nucleobase at the first portion as A and the added nucleobase at the second portion as C.
  • the processor base calls the added nucleobase at the first portion as A and the added nucleobase at the second portion as T.
  • the processor base calls the added nucleobase at the first portion as A and the added nucleobase at the second portion as G.
  • the processor base calls both the added nucleobase at the first portion and the added nucleobase at the second portion as A.
  • T is configured to emit a signal in both the IMAGE 1 channel and the IMAGE 2 channel
  • A is configured to emit a signal in the IMAGE 1 channel only
  • C is configured to emit a signal in the IMAGE 2 channel only
  • G does not emit a signal in either channel.
  • A may be configured to emit a signal in both the IMAGE 1 channel and the IMAGE 2 channel
  • T may be configured to emit a signal in the IMAGE 1 channel only
  • C may be configured to emit a signal in the IMAGE 2 channel only
  • G may be configured to not emit a signal in either channel.
  • Figure 10 is a flow diagram showing a method 1700 of base calling according to the present disclosure.
  • the described method allows for simultaneous sequencing of two (or more) portions (e.g. the first portion and the second portion) in a single sequencing run from a single combined signal obtained from the first portion and the second portion, thus requiring less sequencing reagent consumption and faster generation of data from both the first portion and the second portion.
  • the simplified method may reduce the number of workflow steps while producing the same yield as compared to existing next-generation sequencing methods. Thus, the simplified method may result in reduced sequencing runtime.
  • the disclosed method 1700 may start from block 1701. The method may then move to block 1710.
  • first polynucleotide sequences are extended from the first immobilised primers and second polynucleotide sequences are extended from the second immobilised primers
  • this difference in proportions that are cleavable in the first immobilised primers and the second immobilised primers provides a way of selectively processing the first polynucleotide sequences relative to the second polynucleotide sequences.
  • the second immobilised primer is different in sequence to the first immobilised primer.
  • first immobilised primers i.e. other than the first immobilised primers that are configured to be cleavable under first cleavage conditions
  • the remaining population of first immobilised primers are not cleavable under the first cleavage conditions. As such, only some of the total population of first immobilised primers will become cleaved when the solid support is exposed to the first cleavage conditions.
  • the remaining population of second immobilised primers i.e. other than the second immobilised primers that are configured to be cleavable under second cleavage conditions
  • the remaining population of second immobilised primers are not cleavable under the second cleavage conditions.
  • there is no (or substantially no) remaining population of second immobilised primers As such, some or substantially all of the total population of second immobilised primers will become cleaved when the solid support is exposed to the second cleavage conditions.
  • the phosphate linkage is located at a 5’-end or a 3’-end of a nucleotide comprising an unnatural nucleobase (i.e. one which is not usually present in a typical DNA sequence an RNA sequence).
  • an unnatural nucleobase i.e. one which is not usually present in a typical DNA sequence an RNA sequence.
  • first cleavage conditions refers to reaction conditions that cause cleavage within the first immobilised primer (i.e. at the first cleavage site).
  • the first cleavage conditions may involve exposure to a thermal trigger (e.g. by heating), a light trigger (e.g. by exposure to ultraviolet light), and/or a chemical/biochemical trigger (e.g. an enzyme, a metal catalyst including transition metal catalysts such as a palladiumbased or a nickel-based catalyst, periodate).
  • a thermal trigger e.g. by heating
  • a light trigger e.g. by exposure to ultraviolet light
  • a chemical/biochemical trigger e.g. an enzyme, a metal catalyst including transition metal catalysts such as a palladiumbased or a nickel-based catalyst, periodate.
  • the “first cleavage conditions” may allow linearisation to occur, and may be referred to as “first linearisation conditions”.
  • the proportion of first immobilised primers may be configured to be cleavable by a thermal trigger (e.g. by heating), a light trigger (e.g. by exposure to ultraviolet light), and/or a chemical/biochemical trigger (e.g. an enzyme, a metal catalyst including transition metal catalysts such as a palladium-based or a nickel- based catalyst, periodate).
  • a thermal trigger e.g. by heating
  • a light trigger e.g. by exposure to ultraviolet light
  • a chemical/biochemical trigger e.g. an enzyme, a metal catalyst including transition metal catalysts such as a palladium-based or a nickel- based catalyst, periodate.
  • the proportion of first immobilised primers may be configured to be cleavable by a glycosylase.
  • the first cleavage conditions involve exposure to a glycosylase.
  • the proportion of first immobilised primers may be configured to be cleavable by a glycosylase that recognises any nitrogenous base (e.g. purine or pyrimidine) which is not selected from guanine (G), cytosine (C), adenine (A) and thymine (T) when the first immobilised primer is a DNA sequence; or the proportion of first immobilised primers may be configured to be cleavable by a glycosylase that recognises any nitrogenous base (e.g.
  • the glycosylase may recognise an unnatural nucleobase (i.e. one which is not usually present in a typical DNA sequence an RNA sequence).
  • unnatural nucleobases may include oxoguanine (e.g. 8-oxoguanine), hypoxanthine, xanthine, methylguanines (e.g. O 6 -methylguanine, N 7 - methylguanine), methyladenines (e.g.
  • methylcytosines e.g. 5-methylcytosine, 5-hydroxymethylcytosine, 5- formylcytosine, 5-carboxylcytosine
  • dihydrouracil inosine
  • uracil if the first immobilised primer is a DNA sequence.
  • the proportion of first immobilised primers may be configured to be cleavable by a uracil glycosylase (when the first immobilised primer is a DNA sequence) or an oxoguanine glycosylase (e.g. 8-oxoguanine glycosylase); and in a further embodiment, an oxoguanine glycosylase (e.g. 8-oxoguanine glycosylase).
  • a uracil glycosylase when the first immobilised primer is a DNA sequence
  • an oxoguanine glycosylase e.g. 8-oxoguanine glycosylase
  • an oxoguanine glycosylase e.g. 8-oxoguanine glycosylase
  • each first immobilised primer that is cleavable may comprise a nucleobase which is not selected from guanine, cytosine, adenine or thymine when the first immobilised primer is a DNA sequence, or wherein each first immobilised primer that is cleavable may comprise a nucleobase which is not selected from guanine, cytosine, adenine or uracil when the first immobilised primer is an RNA sequence.
  • each first immobilised primer that is cleavable may comprise an unnatural nucleobase (i.e. one which is not usually present in a typical DNA sequence an RNA sequence).
  • examples of unnatural nucleobases may include oxoguanine (e.g. 8- oxoguanine), hypoxanthine, xanthine, methylguanines (e.g. O 6 -methylguanine, N 7 - methylguanine), methyladenines (e.g. 3-methyladenine, N 6 -methyladenine), modified cytosines including methylcytosines (e.g. 5-methylcytosine, 5-hydroxymethylcytosine, 5- formylcytosine, 5-carboxylcytosine), dihydrouracil, inosine, and uracil (if the first immobilised primer is a DNA sequence).
  • oxoguanine e.g. 8- oxoguanine
  • hypoxanthine e.g. O 6 -methylguanine, N 7 - methylguanine
  • methyladenines e.g. 3-methyladenine, N 6 -methyladenine
  • each first immobilised primer that is cleavable may comprise oxoguanine (e.g. 8-oxoguanine) or uracil when the first immobilised primer is a DNA sequence, or wherein each first immobilised primer that is cleavable may comprise oxoguanine (e.g. 8-oxoguanine) when the first immobilised primer is an RNA sequence; and in an even further embodiment, wherein each first immobilised primer that is cleavable may comprise oxoguanine (e.g. 8-oxoguanine) when the first immobilised primer is a DNA sequence.
  • oxoguanine e.g. 8-oxoguanine
  • uracil when the first immobilised primer is a DNA sequence
  • each first immobilised primer that is cleavable may comprise oxoguanine (e.g. 8-oxoguanine) when the first immobilised primer is an RNA sequence
  • the proportion of second immobilised primers may be configured to be cleavable by a thermal trigger (e.g. by heating), a light trigger (e.g. by exposure to ultraviolet light), and/or a chemical/biochemical trigger (e.g. an enzyme, a metal catalyst including transition metal catalysts such as a palladium-based or a nickel- based catalyst, periodate).
  • a thermal trigger e.g. by heating
  • a light trigger e.g. by exposure to ultraviolet light
  • a chemical/biochemical trigger e.g. an enzyme, a metal catalyst including transition metal catalysts such as a palladium-based or a nickel- based catalyst, periodate.
  • the glycosylase may recognise an unnatural nucleobase (i.e. one which is not usually present in a typical DNA sequence an RNA sequence).
  • unnatural nucleobases may include oxoguanine (e.g. 8-oxoguanine), hypoxanthine, xanthine, methylguanines (e.g. O 6 -methylguanine, N 7 -methylguanine), methyladenines (e.g.
  • the proportion of first immobilised primers cleavable under first cleavage conditions relative to a total population of first immobilised primers may be between 0.2 to 0.8, between 0.25 to 0.75 (in a further embodiment), between 1 Zs to % (in an even further embodiment), or about 0.5 (in a yet even further embodiment); whilst respective proportions of first immobilised primers which are not cleavable under first cleavage conditions relative to a total population of first immobilised primers may be between 0.8 to 0.2, between 0.75 to 0.25 (in the further embodiment), between % to 1 Zs (in the even further embodiment), or about 0.5 (in the yet even further embodiment) (wherein the proportion of first immobilised primers cleavable under first cleavage conditions and the proportion of first immobilised primers which are not cleavable under first cleavage conditions sums to 1).
  • a surface of the solid support may comprise at least one first linking group capable of forming non-covalent interactions, covalent bonds, or metalcoordination bonds with a second linking group; in a further embodiment, non-covalent interactions or covalent bonds.
  • the surface of the solid support may comprise a plurality of first linking groups.
  • first immobilised primer and/or the second immobilised primer may comprise a first linking group capable of forming non-covalent interactions, covalent bonds, or metal-coordination bonds with a second linking group; in a further embodiment, covalent bonds.
  • the first linking groups are advantageous because these can form cross-links with the template strands, thus allowing the template strands to be fixed to the surface of the solid support and/or the immobilised primers, and preventing them from becoming washed away (e.g. during template melting). Although not strictly necessary, this may be useful during amplification, clustering or sequencing of the template, particularly when the first immobilised primer and/or the second immobilised primer is cleaved on exposure to the first cleavage conditions and/or second cleavage conditions.
  • the first linking groups may be located only within the well (or plurality of wells). In other words, a region outside the well (or plurality of wells) may not comprise the first linking groups. In particular, a region outside the well (or plurality of wells) may not comprise the first linking groups, as well as not comprising the first immobilised primers and the second immobilised primers.
  • the first linking groups may be capable of forming non-covalent interactions.
  • These non-covalent interactions may include one or more of ionic bonds, hydrogen bonds, hydrophobic interactions, TT-TT interactions, van der Waals interactions and host-guest interactions.
  • the type of interaction is not particularly limited, provided that the interactions are (collectively) sufficiently strong for the template strands to remain attached to the solid support during amplification, clustering or sequencing.
  • the term “ionic bond” refers to a chemical bond between two or more ions that involves an electrostatic attraction between a cation and an anion.
  • the cation may be selected from “metal cations”, as described herein, or “non-metal cations”.
  • Non-metal cations may include ammonium salts (e.g. alkylammonium salts) or phosphonium salts (e.g. alkylphosphonium salts).
  • the anion may be selected from phosphates, thiophosphates, phosphonates, thiophosphonates, phosphinates, thiophosphinates, sulfates, sulfonates, sulfites, sulfinates, carbonates, carboxylates, alkoxides, phenolates and thiophenolates.
  • hydrogen bond refers to a bonding interaction between a lone pair on an electron-rich atom (e.g. nitrogen, oxygen or fluorine) and a hydrogen atom attached to an electronegative atom (e.g. nitrogen or oxygen).
  • electron-rich atom e.g. nitrogen, oxygen or fluorine
  • hydrogen atom attached to an electronegative atom (e.g. nitrogen or oxygen).
  • the term “host-guest interaction” refers to two or more groups which are able to form bound complexes via one or more types of non-covalent interactions by molecular recognition, such as ionic bonding, hydrogen bonding, hydrophobic interactions, van der Waals interactions and TT-TT interactions.
  • the host- guest interaction may include interactions formed between cucubiturils with adamantanes (e.g. 1-adamantylamine), ammonium ions (e.g. amino acids), ferrocenes; cyclodextrins with adamantanes (e.g. 1-adamantylamine), ammonium ions (e.g.
  • ferrocenes calixarenes with adamantanes (e.g. 1-adamantylamine), ammonium ions (e.g. amino acids), ferrocenes; crown ethers (e.g. 18-crown-6, 15-crown-5, 12- crown-4) or cryptands (e.g. [2.2.2]cryptand) with cations (e.g. metal cations, ammonium ions); avidins (e.g. streptavidin) and biotin; and antibodies and haptens.
  • adamantanes e.g. 1-adamantylamine
  • ammonium ions e.g. amino acids
  • ferrocenes e.g. 18-crown-6, 15-crown-5, 12- crown-4
  • cryptands e.g. [2.2.2]cryptand
  • the first linking groups may be capable of forming covalent bonds.
  • the bond may be stable such that the template strands remain attached to the solid support during amplification, clustering or sequencing.
  • covalent bonds include cycloadducts (e.g. triazole cycloadducts, cyclobutane cycloadducts, furan-maleimide cycloadducts), alkylene linkages, alkenylene linkages, esters, amides, acetals, hemiaminal ethers, aminals, imines, hydrazones, polysulfide linkages (e.g. disulfide linkages), boron-based linkages (e.g. boronic and borinic acids/esters), silicon-based linkages (e.g. silyl ether, siloxane), and phosphorus- based linkages (e.g. phosphite, phosphate, thiophosphate) linkages.
  • cycloadducts e.g. triazole cycloadducts, cyclobutane cycloadducts, furan-maleimide cycloadducts
  • cycloadduct refers to a cyclic structure formed from a cycloaddition reaction between two components (e.g. Diels-Alder type cycloadditions between a diene and a dienophile, including inverse Diels-Alder type cycloadditions, 1 ,3- dipolar type cycloadditions between a dipole and a dipolarophile, or [2 + 2] cycloadditions between two alkenes).
  • Diels-Alder type cycloadditions between a diene and a dienophile including inverse Diels-Alder type cycloadditions, 1 ,3- dipolar type cycloadditions between a dipole and a dipolarophile, or [2 + 2] cycloadditions between two alkenes).
  • alkyl or alkylene refers to monovalent or divalent straight and branched chain groups respectively having from 1 to 12 carbon atoms.
  • the alkyl or alkylene groups are straight or branched alkyl or alkylene groups having from 1 to 6 carbon atoms; in a yet further embodiment, straight or branched alkyl or alkylene groups having from 1 to 4 carbon atoms.
  • An alkyl or alkylene group may comprise one or more “substituents”, as described herein.
  • alkenyl or “alkenylene” refers to monovalent or divalent straight and branched chain groups respectively having from 1 to 12 carbon atoms, and which comprise at least one carbon-carbon double bond.
  • the alkenyl or alkenylene groups are straight or branched alkenyl or alkenylene groups having from 1 to 6 carbon atoms; in a yet further embodiment, straight or branched alkenyl or alkenylene groups having from 1 to 4 carbon atoms.
  • An alkenyl or alkenylene group may comprise one or more “substituents”, as described herein.
  • alkynyl refers to monovalent straight and branched chain groups respectively having from 1 to 12 carbon atoms, and which comprise at least one carbon-carbon triple bond.
  • the alkynyl groups are straight or branched alkynyl groups having from 1 to 6 carbon atoms; in a yet further embodiment, straight or branched alkynyl groups having from 1 to 4 carbon atoms.
  • An alkynyl group may comprise one or more “substituents”, as described herein.
  • amino refers to a -N(R)(R’) group, where R and R’ are independently hydrogen or a “substituent” as defined herein.
  • amine linkage refers to a -NR- group, and where R is hydrogen or a “substituent” as defined herein.
  • aryl refers to a monovalent monocyclic, bicyclic or tricyclic aromatic group respectively containing from 6 to 14 carbon atoms in the ring. Common aryl groups include C6-C14 aryl, for example, Ce-C aryl. An aryl group may comprise one or more “substituents”, as described herein.
  • a “heterocyclyl” group refers to a monovalent saturated or partially saturated 3 to 7 membered monocyclic, or 7 to 10 membered bicyclic ring system respectively, which consists of carbon atoms and from one to four heteroatoms independently selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur heteroatoms may be optionally oxidised, the nitrogen may be optionally quaternised, and includes any bicyclic group in which any of the above-defined rings is fused to a benzene ring, and wherein the ring may be substituted on carbon or on a nitrogen atom if the resulting compound is stable.
  • heterocyclyl groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydrothiopyranyl, isoxazolinyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1 , 2,3,6- tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1 ,3-dioxolanyl, pyrazolinyl, di
  • a heterocyclyl group may comprise one or more “substituents”, as described herein.
  • heteroaryl group refers to monovalent aromatic groups having 5 to 14 ring atoms respectively (for example, 5 to 10 ring atoms) and containing carbon atoms and 1 , 2 or 3 oxygen, nitrogen or sulfur heteroatoms.
  • Non-limiting examples of “heteroaryl” groups include quinolyl including 8-quinolyl, isoquinolyl, coumarinyl including 8-coumarinyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, pyridazinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl (e.g.
  • tetrazolyl isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanylene, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridyl.
  • heteroaryl (or heteroarylene) group contains a nitrogen atom in a ring
  • nitrogen atom may be in the form of an N- oxide, e.g., a pyridyl N-oxide, pyrazinyl N-oxide, pyrimidinyl N-oxide and pyridazinyl N- oxide.
  • a heteroaryl group may comprise one or more “substituents”, as described herein.
  • acetal refers to a -OC(R)(R’)O- group, where R and R’ are independently hydrogen or a “substituent” as described herein.
  • hypothalamic ether refers to a -OC(R)(R’)NR”- group, where R, R’ and R” are independently hydrogen or a “substituent” as described herein.
  • the term “aminal” refers to a -NR(R’)(R”)NR”’- group, where R, R’, R” and R’” are independently hydrogen or a “substituent” as described herein.
  • polysulfide refers to a -(S) n - group, wherein n is 2 to 10, or 2 to 6.
  • n may be 2, forming a “disulfide” linkage.
  • boron-based linkage refers to a -(O) a -B(OR)-(O)b- group, where R is independently hydrogen or a “substituent” as described herein, and where a and b are independently 0 or 1 .
  • silicon-based linkage refers to a -(O) a -Si(R)(R’)-(O)b- group, where R and R’ are independently hydrogen or a “substituent” as described herein, and where a and b are independently 0 or 1 .
  • phosphorus-based linkage refers to a -(O) a -P(R)-(O)b- group, where R and R’ are independently hydrogen or a “substituent” as described herein, and where a and b are independently 0 or 1.
  • substituents may be chosen from the foregoing list.
  • each R’ may be the same or different.
  • the first linking groups may be capable of forming metalcoordination bonds. Where metal-coordination bonds are used, the bond may be strong enough such that the template strands remain attached to the solid support during amplification, clustering or sequencing.
  • the non-terminated first sequencing primers comprise or consist of a sequence selected from SEQ ID NOs: 7 to 10 or a variant or fragment thereof and the second sequencing primers comprises or consists of a different sequence selected from SEQ ID NOs: 7 to 10, or a variant or fragment thereof.
  • the data processing device may comprise a solid support as described herein, such as a flow cell.
  • a computer-readable storage medium comprising instructions which, when executed by a processor, cause the processor to carry out the methods as described herein.
  • a software module can reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of computer-readable storage medium known in the art.
  • An exemplary storage medium can be coupled to the processor such that the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium can be integral to the processor.
  • the processor and the storage medium can reside in an ASIC.
  • a software module can comprise computer-executable instructions which cause a hardware processor to execute the computer-executable instructions.
  • Computer-executable instructions may be stored in a (transitory or non-transitory) computer readable storage medium (e.g., memory, storage system, etc.) storing code, or computer readable instructions.
  • a (transitory or non-transitory) computer readable storage medium e.g., memory, storage system, etc.
  • Disjunctive language such as the phrase “at least one of X, Y or Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to present that an item, term, etc., may be either X, Y or Z, or any combination thereof (e.g., X, Y and/or Z). Thus, such disjunctive language is not generally intended to, and should not, imply that certain embodiments require at least one of X, at least one of Y or at least one of Z to each be present.
  • the terms “about” or “approximate” and the like are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, where the range can be ⁇ 20%, ⁇ 15%, ⁇ 10%, ⁇ 5%, or ⁇ 1%.
  • the term “substantially” is used to indicate that a result (e.g., measurement value) is close to a targeted value, where close can mean, for example, the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.
  • the term “partially” is used to indicate that an effect is only in part or to a limited extent.
  • a device configured to or “a device to” are intended to include one or more recited devices.
  • Such one or more recited devices can also be collectively configured to carry out the stated recitations.
  • a processor to carry out recitations A, B and C can include a first processor configured to carry out recitation A working in conjunction with a second processor configured to carry out recitations B and C.
  • a PAZAM coated, polished, non-grafted HiSeq4K flowcell was grafted with 10 pM P7-8oxoG and 10 M P5-U (lanes 1 and 2): oligo mixes were made up in 1.125M Na2SC>4 with 0.1% Tween20. 175 pl of this buffer was spiked with the appropriate primers and used in the grafting reaction on an Illumina cBot. The grafting mix was pumped onto the flowcell surface and incubated at 60 °C for 60 mins to allow the click chemistry between the 5’ BCNs on the oligos and the free azides to take place.
  • the flowcell lanes were washed with HT 1 buffer and then oligo grafting checked via a TET-QC assay (hybridisation of TET labelled complements to the P5/P7 oligo sequences, Typhoon instrument scanning to assess the levels of TET signal in each lane).
  • TET-QC oligos were removed by 0.1 N NaOH dehyb before the flowcell was used to make clusters.
  • lanes were prepared for either “read 1” or “read 2” by linearising the P5 oligos with LMX1 (for read 1 in lane 1) or linearising the linearizable P7 oligos with FpG (PLM2v2 reagent, HiSeqX PE kit) (for read 2 in lane 2). Linearisation for both cases was for 30 mins at 38 °C. Lanes were then primer hybed with either HP10 (read 1 primer mix) or HP11 (read 2 primer mix) as appropriate.
  • R1 linearised lanes all show similar first cycle intensity (lanes 1 , 3, 5 and 7), while the R2 linearised lanes (lanes 2, 4, 6 and 8) show decreasing intensity due to the increasing concentration of P7-nonlin within the grafting mix.
  • Lanes 3 and 4 in particular show an almost 2: 1 ratio of first cycle intensities as desired, but the flowcell in general shows how the ratio is completely tunable as required.
  • SEQ ID NO. 2 P7 sequence
  • SEQ ID NO. 4 P7’ sequence (complementary to P7)
  • SEQ ID NO. 6 Alternative P5’ sequence (complementary to alternative P5 sequence)
  • SEQ ID NO. 7 SBS3
  • SEQ ID NO. 9 SBS12
  • SEQ ID NO. 12 BCN-P7-8oxoG

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Abstract

Des aspects concernent des supports solides et des procédés destinés à être utilisés dans le séquençage d'acides nucléiques, en particulier des supports solides et des procédés destinés à être utilisés dans le séquençage simultané. L'invention concerne également des procédés et des kits destinés à être utilisés dans le séquençage d'acides nucléiques, en particulier des procédés destinés à être utilisés pour optimiser le rapport signal sur bruit dans un séquençage simultané, en particulier en utilisant une quantité calculée d'amorces à terminaison.
PCT/EP2024/076524 2023-09-20 2024-09-20 Séquençage optimisé d'acides nucléiques WO2025062001A1 (fr)

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