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WO2023175040A2 - Séquençage simultané de brins complémentaires sens et antisens sur des polynucléotides concaténés pour la détection de méthylation - Google Patents

Séquençage simultané de brins complémentaires sens et antisens sur des polynucléotides concaténés pour la détection de méthylation Download PDF

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WO2023175040A2
WO2023175040A2 PCT/EP2023/056668 EP2023056668W WO2023175040A2 WO 2023175040 A2 WO2023175040 A2 WO 2023175040A2 EP 2023056668 W EP2023056668 W EP 2023056668W WO 2023175040 A2 WO2023175040 A2 WO 2023175040A2
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Prior art keywords
sequence
sequencing
complement
signal
polynucleotide
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PCT/EP2023/056668
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English (en)
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WO2023175040A3 (fr
Inventor
Niall Gormley
Jonathan Boutell
Aathavan KARUNAKARAN
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Illumina, Inc.
Illumina Cambridge Limited
Illumina Software, Inc.
Illumina Singapore Pte. Ltd.
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Application filed by Illumina, Inc., Illumina Cambridge Limited, Illumina Software, Inc., Illumina Singapore Pte. Ltd. filed Critical Illumina, Inc.
Priority to AU2023233839A priority Critical patent/AU2023233839A1/en
Priority to CA3223669A priority patent/CA3223669A1/fr
Priority to EP23714638.6A priority patent/EP4341425A2/fr
Publication of WO2023175040A2 publication Critical patent/WO2023175040A2/fr
Publication of WO2023175040A3 publication Critical patent/WO2023175040A3/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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the invention relates to methods of detecting modified cytosines in nucleic acid sequences.
  • cytosines including 5-methylcytosine (5mC)
  • 5mC 5-methylcytosine
  • cfDNA cell-free DNA
  • a method of preparing at least one polynucleotide sequence for detection of modified cytosines comprising: synthesising at least one polynucleotide sequence comprising a first portion and a second portion, wherein the at least one polynucleotide sequence comprises portions of a double-stranded nucleic acid template, and the first portion comprises a forward strand of the template, and the second portion comprises a reverse complement strand of the template; or wherein the first portion comprises a reverse strand of the template, and the second portion comprises a forward complement strand of the template, wherein the template is generated from a target polynucleotide to be sequenced via complementary base pairing, and wherein the target polynucleotide has been pre-treated using a conversion reagent, wherein the conversion reagent is configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil, and/or where
  • the target polynucleotide has been pre-treated using a conversion reagent configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil.
  • the target polynucleotide has been pre-treated using a conversion reagent configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil.
  • the conversion agent comprises a chemical agent and/or an enzyme.
  • the chemical agent comprises a boron-based reducing agent.
  • the boron-based reducing agent is an amine-borane compound or an azine-borane compound.
  • the boron-based reducing agent is selected from the group consisting of pyridine borane, 2-picoline borane, t-butylamine borane, ammonia borane, ethylenediamine borane and dimethylamine borane.
  • the chemical agent comprises sulfite; such as bisulfite or sodium bisulfite.
  • the enzyme comprises a cytidine deaminase.
  • the cytidine deaminase is a wild-type cytidine deaminase or a mutant cytidine deaminase.
  • the cytidine deaminase is a mutant cytidine deaminase.
  • the cytidine deaminase is a member of the AID subfamily, the APOBEC1 subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3B subfamily, the APOBEC3C subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, the APOBEC3G subfamily, the APOBEC3H subfamily, or the APOBEC4 subfamily; in one embodiment, the cytidine deaminase is a member of the APOBEC3A subfamily.
  • the cytidine deaminase comprises amino acid substitution mutations at positions functionally equivalent to (Tyr/Phe)130 and Tyr132 in a wild-type APOBEC3A protein.
  • the (Tyr/Phe)130 is Tyr130
  • the wild-type APOBEC3A protein is SEQ ID NO. 32.
  • substitution mutation at the position functionally equivalent to Tyr130 comprises Ala, Vai or Trp.
  • the substitution mutation at the position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gin or Lys.
  • the mutant cytidine deaminase comprises a ZDD motif H-[P/A/V]-E- X [2 3-28]-P-C-X [2 -4]-C (SEQ ID NO. 67).
  • the mutant cytidine deaminase is a member of the APOBEC3A subfamily and comprises a ZDD motif HXEX 24 SW(S/T)PCX [2 .4]CX6FX8LX5R(L/I)YX [8 - n]LX 2 LX[io]M (SEQ ID NO. 68).
  • the mutant cytidine deaminase converts 5-methylcytosine to thymine by deamination at a greater rate than conversion rate of cytosine to uracil by deamination; wherein the rate may be at least 100-fold greater.
  • the target polynucleotide is treated with a further agent prior to treatment with the conversion reagent.
  • the further agent is configured to convert a modified cytosine to another modified cytosine.
  • the further agent configured to convert a modified cytosine to another modified cytosine comprises a chemical agent and/or an enzyme.
  • the further agent configured to convert a modified cytosine to another modified cytosine comprises an oxidising agent; such as a metal-based oxidising agent; for example, a transition metal-based oxidising agent; or such as a ruthenium- based oxidising agent.
  • an oxidising agent such as a metal-based oxidising agent; for example, a transition metal-based oxidising agent; or such as a ruthenium- based oxidising agent.
  • the further agent configured to convert a modified cytosine to another modified cytosine comprises a reducing agent; such as a Group Ill-based reducing agent; for example, a boron-based reducing agent.
  • the further agent configured to convert a modified cytosine to another modified cytosine comprises a ten-eleven translocation (TET) methylcytosine dioxygenase; for example, wherein the TET methylcytosine dioxygenase may be a member of the TET 1 subfamily, the TET2 subfamily, or the TET3 subfamily.
  • TET ten-eleven translocation
  • the further agent is configured to reduce/prevent deamination of a particular modified cytosine.
  • the further agent configured to reduce/prevent deamination of a particular modified cytosine comprises a chemical agent and/or an enzyme.
  • the further agent configured to reduce/prevent deamination of a particular modified cytosine comprises a glycosyltransferase; such as a p- glucosyltransferase.
  • the further agent configured to reduce/prevent deamination of a particular modified cytosine comprises a hydroxylamine or a hydrazine.
  • the modified cytosine is selected from the group consisting of: 5- methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.
  • the forward strand of the template is not identical to the reverse complement strand of the template.
  • the forward strand comprises a guanine base at a first position, and wherein the reverse complement strand comprises an adenine base at a second position corresponding to the same position number as the first position; or wherein the forward strand comprises an adenine base at a first position, and wherein the reverse complement strand comprises a guanine base at a second position corresponding to the same position number as the first position.
  • the method further comprises a step of preparing the first portion and the second portion for concurrent sequencing.
  • the method comprises simultaneously contacting first sequencing primer binding sites located after a 3’-end of the first portions with first primers and second sequencing primer binding sites located after a 3’-end of the second portions with second primers.
  • a proportion of first portions is capable of generating a first signal and a proportion of second portions is capable of generating a second signal, wherein an intensity of the first signal is substantially the same as an intensity of the second signal.
  • the method further comprises a step of selectively processing the at least one polynucleotide sequence comprising the first portion and the second portion, such that a proportion of first portions are capable of generating a first signal and a proportion of second portions are capable of generating a second signal, wherein the selective processing causes an intensity of the first signal to be greater than an intensity of the second signal.
  • a concentration of the first portions capable of generating the first signal is greater than a concentration of the second portions capable of generating the second signal.
  • a ratio between the concentration of the first portions capable of generating the first signal and the concentration of the second portions capable of generating the second signal is between 1.25:1 to 5:1 , or between 1.5:1 to 3:1 , or about 2:1.
  • selective processing comprises preparing for selective sequencing or conducting selective sequencing.
  • selectively processing comprises contacting first sequencing primer binding sites located after a 3’-end of the first portions with first primers and contacting second sequencing primer binding sites located after a 3’-end of the second portions with second primers, wherein the second primers comprises a mixture of blocked second primers and unblocked second primers.
  • the blocked second primer comprises a blocking group at a 3’ end of the blocked second primer.
  • the blocking group is selected from the group consisting of: a hairpin loop, a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom instead of a 3’-OH group, a phosphate group, a phosphorothioate group, a propyl spacer, a modification blocking the 3’-hydroxyl group, or an inverted nucleobase.
  • the blocked second primer comprises a sequence as defined in SEQ ID NO. 11 to 16 or a variant or fragment thereof and/or the unblocked second primer comprises a sequence as defined in SEQ ID NO. 11 to 14 or a variant or fragment thereof.
  • the first signal and the second signal are spatially unresolved.
  • the at least one polynucleotide sequence comprising the first portion and the second portion is/are attached to a solid support, wherein the solid support may be a flow cell.
  • the at least one polynucleotide sequence comprising the first portion and the second portion forms a cluster on the solid support.
  • the cluster is formed by bridge amplification.
  • the at least one polynucleotide sequence comprising the first portion and the second portion forms a monoclonal cluster.
  • the solid support comprises at least one first immobilised primer and at least one second immobilised primer.
  • the first immobilised primer comprises a sequence as defined in SEQ ID NO. 1 or 5, or a variant or fragment thereof; and the second immobilised primer comprises a sequence as defined in SEQ ID NO. 2, or a variant or fragment thereof.
  • each polynucleotide sequence comprising the first portion and the second portion is attached to a first immobilised primer.
  • each polynucleotide sequence comprising the first portion and the second portion further comprises a second adaptor sequence, wherein the second adaptor sequence is substantially complementary to the second immobilised primer.
  • the step of synthesising the at least one polynucleotide sequence comprising a first portion and a second portion comprises: synthesising a first precursor polynucleotide fragment comprising a complement of the first portion and a hybridisation complement sequence, synthesising a second precursor polynucleotide fragment comprising a second portion and a hybridisation sequence, annealing the hybridisation complement sequence of the first precursor polynucleotide fragment with the hybridisation sequence on the second precursor polynucleotide fragment to form a hybridised adduct, synthesising a first precursor polynucleotide sequence by extending the first precursor polynucleotide fragment to form a complement of the second portion, and synthesising the at least one polynucleotide sequence by forming a complement of the first precursor polynucleotide sequence.
  • the first precursor polynucleotide fragment comprises a first sequencing primer binding site complement.
  • the first sequencing primer binding site complement is located before a 5’-end of the complement of the first portion, such as immediately before the 5’- end of the complement of the first portion.
  • the first precursor polynucleotide fragment comprises a second adaptor complement sequence.
  • the second adaptor complement sequence is located before a 5’- end of the complement of the first portion.
  • the first precursor polynucleotide fragment comprises a first sequencing primer binding site complement and a second adaptor complement sequence.
  • the first sequencing primer binding site complement is located before a 5’-end of the complement of the first portion, and wherein the second adaptor complement sequence is located before a 5’-end of the first sequencing primer binding site complement.
  • the first precursor polynucleotide fragment comprises a second sequencing primer binding site complement.
  • the hybridisation sequence complement comprises the second sequencing primer binding site complement.
  • the second precursor polynucleotide fragment comprises a first adaptor complement sequence.
  • the method further comprises concurrently sequencing nucleobases in the first portion and the second portion.
  • the first portion is at least 25 base pairs and the second portion is at least 25 base pairs.
  • a method of sequencing at least one polynucleotide sequence to detect modified cytosines comprising: preparing at least one polynucleotide sequence for detection of modified cytosines using a method as described herein; concurrently sequencing nucleobases in the first portion and the second portion; and identifying modified cytosines by detecting differences when comparing a sequence output from the first portion with a sequence output from the second portion.
  • the step of concurrently sequencing nucleobases comprises performing sequencing-by-synthesis or sequencing-by-ligation.
  • the step of preparing the at least one polynucleotide sequence comprises using a method as described herein; and wherein the step of concurrent sequencing nucleobases in the first portion and the second portion is based on the intensity of the first signal and the intensity of the second signal.
  • the method further comprises a step of conducting paired-end reads.
  • the step of concurrently sequencing nucleobases comprises:
  • first intensity data comprising a combined intensity of a first signal component obtained based upon a respective first nucleobase at the first portion and a second signal component obtained based upon a respective second nucleobase at the second portion, wherein the first and second signal components are obtained simultaneously;
  • each classification represents a possible combination of respective first and second nucleobases; and (d) based on the selected classification, base calling the respective first and second nucleobases.
  • selecting the classification based on the first and second intensity data comprises selecting the classification based on the combined intensity of the first and second signal components and the combined intensity of the third and fourth signal components.
  • the plurality of classifications comprises sixteen classifications, each classification representing one of sixteen unique combinations of first and second nucleobases.
  • the first signal component, second signal component, third signal component and fourth signal component are generated based on light emissions associated with the respective nucleobase.
  • the light emissions are detected by a sensor, wherein the sensor is configured to provide a single output based upon the first and second signals.
  • the senor comprises a single sensing element.
  • the method further comprises repeating steps (a) to (d) for each of a plurality of base calling cycles.
  • the step of concurrently sequencing nucleobases comprises:
  • first intensity data comprising a combined intensity of a first signal component obtained based upon a respective first nucleobase at the first portion and a second signal component obtained based upon a respective second nucleobase at the second portion, wherein the first and second signal components are obtained simultaneously;
  • selecting the classification based on the first and second intensity data comprises selecting the classification based on the combined intensity of the first and second signal components and the combined intensity of the third and fourth signal components.
  • an intensity of the first signal component is substantially the same as an intensity of the second signal component and an intensity of the third signal component is substantially the same as an intensity of the fourth signal component.
  • the plurality of classifications consists of a predetermined number of classifications.
  • the plurality of classifications comprises: one or more classifications representing matching first and second nucleobases; and one or more classifications representing mismatching first and second nucleobases
  • determining sequence information of the first portion and second portion comprises: in response to selecting a classification representing matching first and second nucleobases, determining a match between the first and second nucleobases; or in response to selecting a classification representing mismatching first and second nucleobases, determining a mismatch between the first and second nucleobases.
  • determining sequence information of the first portion and the second portion comprises, in response to selecting a classification representing a match between the first and second nucleobases, base calling the first and second nucleobases.
  • determining sequence information of the first portion and the second portion comprises, based on the selected classification, determining that the second portion is modified relative to the first portion at a location associated with the first and second nucleobases.
  • the first signal component, second signal component, third signal component and fourth signal component are generated based on light emissions associated with the respective nucleobase.
  • the light emissions are detected by a sensor, wherein the sensor is configured to provide a single output based upon the first and second signals.
  • the senor comprises a single sensing element.
  • the method further comprises repeating steps (a) to (d) for each of a plurality of base calling cycles.
  • kits comprising instructions for preparing at least one polynucleotide sequence for detection of modified cytosines as described herein, and/or for sequencing at least one polynucleotide sequence to detect modified cytosines as described herein.
  • a data processing device comprising means for carrying out a method as described herein.
  • the data processing device is a polynucleotide sequencer.
  • a computer program product comprising instructions which, when the program is executed by a processor, cause the processor to carry out a method as described herein.
  • a computer- readable storage medium comprising instructions which, when executed by a processor, cause the processor to carry out a method as described herein.
  • a computer- readable data carrier having stored thereon a computer program product as described herein.
  • Figure 1 shows a forward strand, reverse strand, forward complement strand, and reverse complement strand of a polynucleotide molecule.
  • Figure 2 shows the preparation of a concatenated polynucleotide sequence comprising a first portion and a second portion using a tandem insert method, comprising (A) preparation of a desired first (forked) adaptor and second (forked) adaptor from three oligos; (B) different types of first (forked) adaptors and second (forked) adaptors that do not anneal to each other due to the presence of a third oligo on at least one of the first (forked) adaptor and/or the second (forked) adaptor; (C) ligation of the template polynucleotide strand and adaptors generates three products, with the desired product containing both types of adaptor being produced at a proportion of 50%; (D) synthesis of concatenated strands from the desired product; and (E) completion of the synthesis of the concatenated strands from the desired product.
  • A preparation of a desired first (forked) adaptor and second (forked) adaptor from three oligo
  • Figure 3 shows an example of a concatenated polynucleotide sequence comprising a first portion and a second portion, as well as terminal and internal adaptor sequences.
  • Figure 4 shows an example of a concatenated polynucleotide sequence comprising a first portion and a second portion, as well as terminal and internal adaptor sequences.
  • Figure 5 shows a typical solid support.
  • Figure 6 shows the stages of bridge amplification for concatenated polynucleotide sequences and the generation of an amplified cluster, comprising (A) a concatenated library strand hybridising to a immobilised primer; (B) generation of a template strand from the library strand; (C) dehybridisation and washing away the library strand; (D) generation of a template complement strand from the template strand via bridge amplification and dehybridisation of the sequence bridge; (E) further amplification to provide a plurality of template and template complement strands; and (F) cleavage of one set of the template and template complement strands.
  • Figure 7 shows the detection of nucleobases using 4-channel, 2-channel and 1 -channel chemistry.
  • Figure 8 shows a method of selective sequencing.
  • Figure 9 is a plot showing graphical representations of sixteen distributions of signals generated by polynucleotide sequences according to one embodiment.
  • Figure 10 is a flow diagram showing a method for base calling according to one embodiment.
  • Figure 11 is a plot showing graphical representations of nine distributions of signals generated by polynucleotide sequences according to one embodiment.
  • Figure 12 shows the effect of unmodified cytosine to uracil conversion treatment of a double-stranded polynucleotide, and a scatter plot showing the resulting distributions of signals generated by polynucleotide sequences.
  • Figure 13 shows the effect of modified cytosine to thymine conversion treatment of a double-stranded polynucleotide, and a scatter plot showing the resulting distributions of signals generated by polynucleotide sequences.
  • Figure 14 shows alternative signal distributions using a different dye-encoding scheme.
  • Figure 15 shows alternative signal distributions using a different dye-encoding scheme.
  • Figure 16 shows alternative signal distributions using a different dye-encoding scheme.
  • Figure 17 is a flow diagram showing a method for determining sequence information according to one embodiment.
  • Figure 18A shows the effect of pre-treatment of library strands using C to II conversion on bases in template strands.
  • Figure 18B shows the effect of pre-treatment of library strands using mC to T conversion on bases in template strands.
  • Figure 19A shows 9 QaM analysis conducted on the signals obtained from the custom second hyb run of Example 1 .
  • the x-axis shows signal intensity from a “red” wavelength channel, whilst the y-axis shows signal intensity from a “green” wavelength channel.
  • G is not associated with any dyes and as such appears contributes no intensity for both “red” and “green” channels.
  • C is associated with a “red” dye and as such contributes intensity to the “red” channel, but not the “green” channel.
  • T is associated with a “green” dye and as such contributes intensity to the “green” channel, but not the “red channel.
  • A is associated with both a “red” dye and a “green” dye, and as such contributes intensity to both the “red” channel and “green” channel. Since the template comprises forward and reverse complement strands that are sequenced simultaneously, most of the readout will generate (G,G) read (bottom left corner), (C,C) read (bottom right corner), (T,T) read (top left corner), and (A, A) read (top right corner) clouds. However, a central cloud corresponding to (C,T) or (T,C) reads corresponds with the presence of modified cytosines.
  • Figure 19B shows sequence data generated from two different primers used (HYB2’-ME and HP10) in the custom second hyb run of Example 1.
  • the present invention can be used in sequencing, in particular concurrent sequencing. Methodologies applicable to the present invention have been described in WO 08/041002, WO 07/052006, WO 98/44151 , WO 00/18957, WO 02/06456, WO 07/107710, WO05/068656, US 13/661 ,524 and US 2012/0316086, the contents of which are herein incorporated by reference.
  • variant refers to a variant polypeptide sequence or part of the polypeptide sequence that retains desired function of the full non-variant sequence.
  • a desired function of the immobilised primer retains the ability to bind (i.e. hybridise) to a target sequence.
  • a “variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%,
  • sequence identity of a variant can be determined using any number of sequence alignment programs known in the art.
  • fragment refers to a functionally active series of consecutive nucleic acids from a longer nucleic acid sequence.
  • the fragment may be at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 30% the length of the longer nucleic acid sequence.
  • a fragment as used herein may also retain the ability to bind (i.e. hybridise) to a target sequence.
  • Sequencing generally comprises four fundamental steps: 1) library preparation to form a plurality of target polynucleotides for identification; 2) cluster generation to form an array of amplified template polynucleotides; 3) sequencing the cluster array of amplified template polynucleotides; and 4) data analysis to identify characteristics of the target polynucleotides from the amplified template polynucleotide sequences.
  • the polynucleotide sequence 100 comprises a forward strand of the sequence 101 and a reverse strand of the sequence 102. See Figure 1.
  • replication of the polynucleotide sequence 100 provides a double-stranded polynucleotide sequence 100a that comprises a forward strand of the sequence 101 and a forward complement strand of the sequence 10T, and a double-stranded polynucleotide sequence 100b that comprises a reverse strand of the sequence 102 and a reverse complement strand of the sequence 102’.
  • the term “template” may be used to describe a complementary version of the double- stranded polynucleotide sequence 100.
  • the “template” comprises a forward complement strand of the sequence 10T and a reverse complement strand of the sequence 102’.
  • a sequencing process e.g. a sequencing- by-synthesis or a sequencing-by-ligation process
  • reproduces information that was present in the original forward strand of the sequence 101 by using the reverse complement strand of the sequence 102’ as a template for complementary base pairing, a sequencing process (e.g. a sequencing-by-synthesis or a sequencing-by-ligation process) reproduces information that was present in the original reverse strand of the sequence 102.
  • the two strands in the template may also be referred to as a forward strand of the template 10T and a reverse strand of the template 102’.
  • the complement of the forward strand of the template 10T is termed the forward complement strand of the template 101
  • the complement of the reverse strand of the template 102’ is termed the reverse complement strand of the template 102.
  • forward strand, reverse strand, forward complement strand, and reverse complement strand are used herein without qualifying whether they are with respect to the original polynucleotide sequence 100 or with respect to the “template”, these terms may be interpreted as referring to the “template”.
  • Library preparation is the first step in any high-throughput sequencing platform. These libraries allow templates to be generated via complementary base pairing that can subsequently be clustered and amplified. During library preparation, nucleic acid sequences, for example genomic DNA sample, or cDNA or RNA sample, is converted into a sequencing library, which can then be sequenced.
  • the first step in library preparation is random fragmentation of the DNA sample. Sample DNA is first fragmented and the fragments of a specific size (typically 200-500 bp, but can be larger) are ligated, sub-cloned or “inserted” in-between two oligo adaptors (adaptor sequences). The original sample DNA fragments are referred to as “inserts”.
  • the target polynucleotides may advantageously also be size-fractionated prior to modification with the adaptor sequences.
  • the templates to be generated from the libraries may include a concatenated polynucleotide sequence comprising a first portion and a second portion. Generating these templates from particular libraries may be performed according to methods known to persons of skill in the art. However, some example approaches of preparing libraries suitable for generation of such templates are described below.
  • the library may be prepared by using a tandem insert method described in more detail in e.g. WO 2022/087150, which is incorporated herein by reference. This procedure may be used, for example, for preparing templates comprising concatenated polynucleotide sequences comprising a first portion and a second portion, wherein the first portion is a forward strand of the template, and the second portion is a reverse complement strand of the template (or alternatively, wherein the first portion is a reverse strand of the template, and the second portion is a forward complement strand of the template).
  • Such libraries may also be referred to as cross-tandem inserts.
  • a representative process for conducting a tandem insert method is shown in Figure 2A to 2E.
  • one strand of a concatenated polynucleotide within a polynucleotide library may comprise, in a 5’ to 3’ direction, a second primer-binding complement sequence 302 (e.g. P7), a first terminal sequencing primer binding site complement 303’ (e.g. B15-ME; or if ME is not present, then B15), a first insert sequence 401 , a hybridisation complement sequence 403 (e.g. ME’-HYB2-ME; or if ME’ and ME are not present, then HYB2), a second insert sequence 402, a second terminal sequencing primer binding site 304 (e.g. ME’-A14’; or if ME’ is not present, then A14’), and a first primer-binding sequence 301’ (e.g. P5’) ( Figures 3 and 4 - bottom strand).
  • a second primer-binding complement sequence 302 e.g. P7
  • a first terminal sequencing primer binding site complement 303’ e.
  • the strand may further comprise one or more index sequences.
  • a first index sequence (e.g. i7) may be provided between the second primer-binding complement sequence 302 (e.g. P7) and the first terminal sequencing primer binding site complement 303’ (e.g. B15-ME; or if ME is not present, then B15).
  • a second index complement sequence (e.g. i5’) may be provided between the second terminal sequencing primer binding site 304 (e.g. ME’-A14’) and the first primer-binding sequence 30T (e.g. P5’).
  • one strand of a polynucleotide within a polynucleotide library may comprise, in a 5’ to 3’ direction, a second primer-binding complement sequence 302 (e.g. P7), a first index sequence (e.g. i7), a first terminal sequencing primer binding site complement 303’ (e.g. B15-ME; or if ME is not present, then B15), a first insert sequence 401 , a hybridisation complement sequence 403 (e.g. ME’-HYB2-ME; or if ME’ and ME are not present, then HYB2), a second insert sequence 402, a second terminal sequencing primer binding site 304 (e.g. ME’-A14’; or if ME’ is not present, then A14’), a second index complement sequence (e.g. i5’), and a first primer-binding sequence 30T (e.g. P5’)
  • a second primer-binding complement sequence 302 e.g. P7
  • Another strand of a concatenated polynucleotide within a polynucleotide library may comprise, in a 5’ to 3’ direction, a first primer-binding complement sequence 301 (e.g. P5), a second terminal sequencing primer binding site complement 304’ (e.g. A14-ME; or if ME is not present, then A14), a second insert complement sequence 402’, a hybridisation sequence 403’ (e.g. ME’-HYB2’-ME; or if ME’ and ME are not present, then HYB2’), a first insert complement sequence 40T, a first terminal sequencing primer binding site 303 (e.g. ME’-B15’; or if ME’ is not present, then B15’), and a second primer- binding sequence 302’ (e.g. P7’) ( Figures 3 and 4 - top strand).
  • a first primer-binding complement sequence 301 e.g. P5
  • the another strand may further comprise one or more index sequences.
  • a second index sequence (e.g. i5) may be provided between the first primer-binding complement sequence 301 (e.g. P5) and the second terminal sequencing primer binding site complement 304’ (e.g. A14-ME; or if ME is not present, then A14).
  • a first index complement sequence (e.g. i7’) may be provided between the first terminal sequencing primer binding site 303 (e.g. ME’-B15’; or if ME’ is not present, then B15’) and the second primer-binding sequence 302’ (e.g. P7’).
  • another strand of a polynucleotide within a polynucleotide library may comprise, in a 5’ to 3’ direction, a first primer-binding complement sequence 301 (e.g. P5), a second index sequence (e.g. i5), a second terminal sequencing primer binding site complement 304’ (e.g. A14-ME; or if ME is not present, then A14).), a second insert complement sequence 402’, a hybridisation sequence 403’ (e.g. ME’-HYB2’-ME; or if ME’ and ME are not present, then HYB2’), a first insert complement sequence 40T, a first terminal sequencing primer binding site 303 (e.g. ME’-B15’; or if ME’ is not present, then B15’), a first index complement sequence (e.g. i7’), and a second primer-binding sequence 302’ (e.g. P7’).
  • a first primer-binding complement sequence 301 e.
  • the first insert sequence 401 and the second insert sequence 402 may comprise different types of library sequences.
  • the first insert sequence 401 may comprise a forward strand of the sequence 101
  • the second insert sequence may comprise a reverse complement strand of the sequence 102’ (or the first insert sequence 401 may comprise a reverse strand of the sequence 102, and the second insert sequence 402 may comprise a forward complement strand of the sequence 10T), for example where the library is prepared using a tandem insert method.
  • a double-stranded nucleic acid will typically be formed from two complementary polynucleotide strands comprised of deoxyribonucleotides or ribonucleotides joined by phosphodiester bonds, but may additionally include one or more ribonucleotides and/or non-nucleotide chemical moieties and/or non-naturally occurring nucleotides and/or non-naturally occurring backbone linkages.
  • the double-stranded nucleic acid may include non- nucleotide chemical moieties, e.g. linkers or spacers, at the 5' end of one or both strands.
  • the double-stranded nucleic acid may include methylated nucleotides, uracil bases, phosphorothioate groups, peptide conjugates etc.
  • Such non-DNA or non-natural modifications may be included in order to confer some desirable property to the nucleic acid, for example to enable covalent, non-covalent or metal-coordination attachment to a solid support, or to act as spacers to position the site of cleavage an optimal distance from the solid support.
  • a single stranded nucleic acid consists of one such polynucleotide strand.
  • a polynucleotide strand is only partially hybridised to a complementary strand - for example, a long polynucleotide strand hybridised to a short nucleotide primer - it may still be referred to herein as a single stranded nucleic acid.
  • a sequence comprising at least a primer-binding sequence (a primer-binding sequence and a sequencing primer binding site, or a combination of a primer-binding sequence, an index sequence and a sequencing primer binding site) may be referred to herein as an adaptor sequence, and an insert (or inserts in concatenated strands) is flanked by a 5’ adaptor sequence and a 3’ adaptor sequence.
  • the primer-binding sequence may also comprise a sequencing primer for the index read.
  • an “adaptor” refers to a sequence that comprises a short sequence- specific oligonucleotide that is ligated to the 5' and 3' ends of each DNA (or RNA) fragment in a sequencing library as part of library preparation.
  • the adaptor sequence may further comprise non-peptide linkers.
  • the P5’ and P7’ primer-binding sequences are complementary to short primer sequences (or lawn primers) present on the surface of a flow cell. Binding of P5’ and P7’ to their complements (P5 and P7) on - for example - the surface of the flow cell, permits nucleic acid amplification. As used herein denotes the complementary strand.
  • the primer-binding sequences in the adaptor which permit hybridisation to amplification primers will typically be around 20-40 nucleotides in length, although the invention is not limited to sequences of this length.
  • the precise identity of the amplification primers (e.g. lawn primers), and hence the cognate sequences in the adaptors, are generally not material to the invention, as long as the primer-binding sequences are able to interact with the amplification primers in order to direct PCR amplification.
  • sequence of the amplification primers may be specific for a particular target nucleic acid that it is desired to amplify, but in other embodiments these sequences may be "universal" primer sequences which enable amplification of any target nucleic acid of known or unknown sequence which has been modified to enable amplification with the universal primers.
  • the criteria for design of PCR primers are generally well known to those of ordinary skill in the art.
  • the index sequences are unique short DNA (or RNA) sequences that are added to each DNA (or RNA) fragment during library preparation.
  • the unique sequences allow many libraries to be pooled together and sequenced simultaneously. Sequencing reads from pooled libraries are identified and sorted computationally, based on their barcodes, before final data analysis. Library multiplexing is also a useful technique when working with small genomes or targeting genomic regions of interest. Multiplexing with barcodes can exponentially increase the number of samples analysed in a single run, without drastically increasing run cost or run time. Examples of tag sequences are found in WO05/068656, whose contents are incorporated herein by reference in their entirety.
  • the tag can be read at the end of the first read, or equally at the end of the second read, for example using a sequencing primer complementary to the strand marked P7.
  • the invention is not limited by the number of reads per cluster, for example two reads per cluster: three or more reads per cluster are obtainable simply by dehybridising a first extended sequencing primer, and rehybridising a second primer before or after a cluster repopulation/strand resynthesis step. Methods of preparing suitable samples for indexing are described in, for example WO 2008/093098, which is incorporated herein by reference. Single or dual indexing may also be used. With single indexing, up to 48 unique 6-base indexes can be used to generate up to 48 uniquely tagged libraries.
  • up to 24 unique 8-base Index 1 sequences and up to 16 unique 8-base Index 2 sequences can be used in combination to generate up to 384 uniquely tagged libraries. Pairs of indexes can also be used such that every i5 index and every i7 index are used only one time. With these unique dual indexes, it is possible to identify and filter indexed hopped reads, providing even higher confidence in multiplexed samples.
  • the sequencing primer binding sites are sequencing and/or index primer binding sites and indicate the starting point of the sequencing read.
  • a sequencing primer anneals (i.e. hybridises) to at least a portion of the sequencing primer binding site on the template strand.
  • the polymerase enzyme binds to this site and incorporates complementary nucleotides base by base into the growing opposite strand.
  • the hybridisation sequence may comprise an internal sequencing primer binding site.
  • an internal sequencing primer binding site may form part of the hybridisation sequence.
  • ME’-HYB2 (or ME’-HYB2’) may act as an internal sequencing primer binding site to which a sequencing primer can bind.
  • the hybridisation sequence may be an internal sequencing primer binding site.
  • HYB2 (or HYB2’) may act as an internal sequencing primer binding site to which a sequencing primer can bind. Accordingly, we may refer to the hybridisation site herein as comprising a second sequencing primer binding site, or as a second sequencing primer binding site.
  • the target polynucleotide (or in some embodiments, the polynucleotide library) is pre- treated to allow sequencing of modified cytosines. Such methods are described in further detail herein.
  • a double stranded nucleic acid library is formed, typically, the library has previously been subjected to denaturing conditions to provide single stranded nucleic acids. Suitable denaturing conditions will be apparent to the skilled reader with reference to standard molecular biology protocols (Sambrook et al., 2001 , Molecular Cloning, A Laboratory Manual, 4th Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, NY; Current Protocols, eds Ausubel et al). In one embodiment, chemical denaturation may be used.
  • a single-stranded library may be contacted in free solution onto a solid support comprising surface capture moieties (for example P5 and P7 lawn primers).
  • surface capture moieties for example P5 and P7 lawn primers.
  • embodiments of the present invention may be performed on a solid support 200, such as a flowcell.
  • seeding and clustering can be conducted off-flowcell using other types of solid support.
  • the solid support 200 may comprise a substrate 204. See Figure 5.
  • the substrate 204 comprises at least one well 203 (e.g. a nanowell), and typically comprises a plurality of wells 203 (e.g. a plurality of nanowells).
  • the solid support comprises at least one first immobilised primer and at least one second immobilised primer.
  • each well 203 may comprise at least one first immobilised primer 201 , and typically may comprise a plurality of first immobilised primers 201.
  • each well 203 may comprise at least one second immobilised primer 202, and typically may comprise a plurality of second immobilised primers 202.
  • each well 203 may comprise at least one first immobilised primer 201 and at least one second immobilised primer 202, and typically may comprise a plurality of first immobilised primers 201 and a plurality of second immobilised primers 202.
  • the first immobilised primer 201 may be attached via a 5’-end of its polynucleotide chain to the solid support 200.
  • the extension may be in a direction away from the solid support 200.
  • the second immobilised primer 202 may be attached via a 5’-end of its polynucleotide chain to the solid support 200.
  • the extension may be in a direction away from the solid support 200.
  • the first immobilised primer 201 may be different to the second immobilised primer 202 and/or a complement of the second immobilised primer 202.
  • the second immobilised primer 202 may be different to the first immobilised primer 201 and/or a complement of the first immobilised primer 201.
  • the (or each of the) first immobilised primer(s) 201 may comprise a sequence as defined in SEQ ID NO. 1 or 5, or a variant or fragment thereof.
  • the second immobilised primer(s) 202 may comprise a sequence as defined in SEQ ID NO. 2, or a variant or fragment thereof.
  • the solid support may be contacted with the template to be amplified under conditions which permit hybridisation (or annealing - such terms may be used interchangeably) between the template and the immobilised primers.
  • the template is usually added in free solution under suitable hybridisation conditions, which will be apparent to the skilled reader.
  • hybridisation conditions are, for example, 5xSSC at 40°C.
  • other temperatures may be used during hybridisation, for example about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C. Solid-phase amplification can then proceed.
  • the first step of the amplification is a primer extension step in which nucleotides are added to the 3' end of the immobilised primer using the template to produce a fully extended complementary strand.
  • the template is then typically washed off the solid support.
  • the complementary strand will include at its 3' end a primer-binding sequence (i.e. either P5’ or P7’) which is capable of bridging to the second primer molecule immobilised on the solid support and binding.
  • Further rounds of amplification leads to the formation of clusters or colonies of template molecules bound to the solid support. This is called clustering.
  • amplification may be isothermal amplification using a strand displacement polymerase; or may be exclusion amplification as described in WO 2013/188582. Further information on amplification can be found in WO 02/06456 and WO 07/107710, the contents of which are incorporated herein in their entirety by reference.
  • a cluster of template molecules comprising copies of a template strand and copies of the complement of the template strand.
  • one set of strands may be removed from the solid support leaving either the original template strands or the complement strands. Suitable methods for removing such strands are described in more detail in application number WO 07/010251 , the contents of which are incorporated herein by reference in their entirety.
  • each polynucleotide sequence may be attached (via the 5’-end of the (concatenated) polynucleotide sequence) to a first immobilised primer.
  • Each polynucleotide sequence may comprise a second adaptor sequence, wherein the second adaptor comprises a portion which is substantially complementary to the second immobilised primer (or is substantially complementary to the second immobilised primer).
  • the second adaptor sequence may be at a 3’-end of the (concatenated) polynucleotide sequence.
  • a solution comprising a polynucleotide library prepared by a tandem insert method as described above may be flowed across a flowcell.
  • a particular concatenated 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 sequencing primer binding site complement 303’ (e.g. B15-ME), a first insert sequence 401 , a hybridisation complement sequence 403 (e.g. ME’-HYB2-ME), a second insert sequence 402, a second terminal sequencing primer binding site 304 (e.g. ME’-A14’), and a first primer-binding sequence 30T (e.g. P5’), may anneal (via the first primer-binding sequence 30T) to the first immobilised primer 201 (e.g. P5 lawn primer) located within a particular well 203 ( Figure 6A).
  • a second primer-binding complement sequence 302 e.g. P7
  • a first terminal sequencing primer binding site complement 303’ e.g. B15-ME
  • the polynucleotide library may comprise other concatenated polynucleotide strands with different first insert sequences 401 and second insert sequences 402. 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 concatenated 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.
  • the first immobilised primer 201 e.g. P5 lawn primer
  • a second terminal sequencing primer binding site complement 304 e.g. A14-ME; or if ME is not present, then A14
  • a second insert complement sequence 402’ which represents a type of “second portion”
  • a hybridisation sequence 403’ which comprises a type of “second sequencing primer binding site”
  • a first insert complement sequence 40T (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”)
  • a second primer-binding sequence 302 (e.g. P7’) ( Figure 6B).
  • a polymerase such as a DNA or RNA polymerase. If the polynucleotides in the library comprise index sequences, then corresponding index sequences are also produced in the template.
  • the concatenated 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 6C).
  • 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”.
  • a second immobilised primer 202 e.g. P7 lawn primer
  • a new polynucleotide strand may then be synthesised by bridge amplification, extending from the second immobilised primer 202 (e.g. P7 lawn primer) (initially) in a direction away from the substrate 204.
  • the second immobilised primer 202 e.g. P7 lawn primer
  • a first terminal sequencing primer binding site complement 303’ e.g. B15-ME; or if ME is not present, then B15
  • a first insert sequence 401 e.g.
  • a polymerase such as a DNA or RNA polymerase.
  • 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 6D).
  • 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’) on the template strand attached to the first immobilised primer 201 (e.g. P5 lawn primer) may then anneal to another second immobilised primer 202 (e.g. P7 lawn primer) located within the well 203.
  • the first primer- binding sequence 30T (e.g. P5’) on the template strand attached to the second immobilised primer 202 (e.g.
  • P7 lawn primer may then anneal to another first immobilised primer 201 (e.g. P5 lawn primer) located within the well 203.
  • Completion of bridge amplification and dehybridisation may then provide an amplified cluster, thus providing a plurality of concatenated polynucleotide sequences comprising a first insert complement sequence 401’ (i.e. “first portions”) and a second insert complement sequence 402’ (i.e. second portions”), as well as a plurality of concatenated polynucleotide sequences comprising a first insert sequence 401 and a second insert sequence 402 ( Figure 6E).
  • one group of strands (either the group of template polynucleotides, or the group of template complement polynucleotides thereof) is removed from the solid support to form a (monoclonal) cluster, leaving either the templates or the template complements ( Figure 6F).
  • 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
  • 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 7 - 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.
  • a fourth nucleotide type may include a fourth label (e.g. configured to emit a fourth wavelength, such as yellow light).
  • Four images can then be obtained, each using a detection channel that is selective for one of the four different labels.
  • the first nucleotide type e.g. A
  • the second nucleotide type e.g. G
  • the second channel e.g. configured to detect the second wavelength, such as blue light
  • the third nucleotide type e.g. T
  • a third channel e.g.
  • the fourth nucleotide type (e.g. C) may be detected in a fourth channel (e.g. configured to detect the fourth wavelength, such as yellow light).
  • a fourth channel e.g. configured to detect the fourth wavelength, such as yellow light.
  • detection of each nucleotide type may be conducted using fewer than four different labels.
  • sequencing-by-synthesis may be performed using methods and systems described in US 2013/0079232, which is incorporated herein by reference.
  • two channels may be used to detect four nucleobases (also known as 2-channel chemistry) ( Figure 7 - middle).
  • a first nucleotide type e.g. A
  • a second label e.g. configured to emit a second wavelength, such as red light
  • a second nucleotide type e.g. G
  • a third nucleotide type e.g. T
  • the first label e.g.
  • the first nucleotide type (e.g. A) may be detected in both a first channel (e.g. configured to detect the first wavelength, such as red light) and a second channel (e.g. configured to detect the second wavelength, such as green light), the second nucleotide type (e.g.
  • the third nucleotide type (e.g. T) may be detected in the first channel (e.g. configured to detect the first wavelength, such as red light) and may not be detected in the second channel
  • the fourth nucleotide type (e.g. C) may not be detected in the first channel and may be detected in the second channel (e.g. configured to detect the second wavelength, such as green light).
  • one channel may be used to detect four nucleobases (also known as 1 -channel chemistry) ( Figure 7 - 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.
  • a first image can then be obtained, and a subsequent treatment carried out to cleave the label attached to the first nucleotide type, and to attach the label to the label-accepting site on the fourth nucleotide type.
  • a second image may then be obtained.
  • the first nucleotide type e.g. A
  • the second nucleotide type e.g. G
  • the third nucleotide type e.g. T
  • the channel e.g.
  • the fourth nucleotide type (e.g. C) may not be detected in the channel in the first image and may be detected in the channel in the second image (e.g. configured to detect the wavelength, such as green light).
  • 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 sequencing read may comprise the binding of a first sequencing primer (also known as a read 1 sequencing primer) to the first sequencing primer binding site (e.g. first terminal sequencing primer binding site 303 in templates including a concatenated polynucleotide sequence comprising a first portion and a second portion).
  • the second sequencing read may comprise the binding of a second sequencing primer (also known as a read 2 sequencing primer) to the second sequencing primer binding site (e.g. a portion of hybridisation sequence 403’ in templates including a concatenated polynucleotide sequence comprising a first portion and a second portion).
  • first insert complement sequence 40T in templates including a concatenated polynucleotide sequence comprising a first portion and a second portion
  • second insert complement sequence 402 in templates including a concatenated polynucleotide sequence comprising a first portion and a second portion
  • sequencing by ligation for example as described in US 6,306,597 or WO 06/084132, the contents of which are incorporated herein by reference.
  • methods for sequencing described above generally relate to conducting non- selective sequencing.
  • methods of the present invention relating to selective processing may comprise conducting selective sequencing, which is described in further detail below under selective processing.
  • selective processing methods may be used to generate signals of different intensities.
  • the method may comprise selectively processing at least one polynucleotide sequence comprising a first portion and a second portion, such that a proportion of first portions are capable of generating a first signal and a proportion of second portions are capable of generating a second signal, wherein the selective processing causes an intensity of the first signal to be greater than an intensity of the second signal.
  • the method may comprise selectively processing a plurality of polynucleotide sequences each comprising a first portion and a second portion, such that a proportion of first portions are capable of generating a first signal and a proportion of second portions are capable of generating a second signal, wherein the selective processing causes an intensity of the first signal to be greater than an intensity of the second signal.
  • selective processing is meant here performing an action that changes relative properties of the first portion and the second portion in the at least one polynucleotide sequence comprising a first portion and a second portion (or the plurality of polynucleotide sequences each comprising a first portion and a second portion), so that the intensity of the first signal is greater than the intensity of the second signal.
  • the property may be, for example, a concentration of first portions capable of generating the first signal relative to a concentration of second portions capable of generating the second signal.
  • the action may include, for example, conducting selective sequencing, or preparing for selective sequencing.
  • the selective processing results in the concentration of the first portions capable of generating the first signal being greater than the concentration of the second portions capable of generating the second signal.
  • the method of the invention results in an altered ratio of R1 :R2 molecules, such as within a single cluster or a single well.
  • the ratio may be between 1.25:1 to 5:1 , between 1.5:1 to 3:1 , or about 2:1.
  • Selective processing may refer to conducting selective sequencing.
  • selective processing may refer to preparing for selective sequencing.
  • selective sequencing may be achieved using a mixture of unblocked and blocked sequencing primers.
  • the single (concatenated) polynucleotide strand may comprise a first sequencing primer binding site and a second sequencing primer binding site, 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.
  • binding of first sequencing primers to the first sequencing primer site generates a first signal and binding of second sequencing primers to the second sequencing primer site generates a second signal, where the intensity of the first signal is greater than the intensity of the second signal.
  • binding of first sequencing primers to the first sequencing primer site generates a first signal and binding of second sequencing primers to the second sequencing primer site generates a second signal, where the intensity of the first signal is greater than the intensity of the second signal.
  • any ratio of blocked:unblocked second primers can be used that generates a second signal that is of a lower intensity than the first signal, for example, the ratio of blocked:unblocked primers may be: 20:80 to 80:20, or 1 :2 to 2:1.
  • a ratio of 50:50 of blocked: unblocked second primers is used, which in turn generates a second signal that is around 50% of the intensity of the first signal.
  • the first and second sequencing primers may be added to the flow cell at the same time, or separately but sequentially.
  • blocking groups include a hairpin loop (e.g. a polynucleotide attached to the 3’-end, comprising in a 5’ to 3’ direction, a cleavable site such as a nucleotide comprising uracil, a loop portion, and a complement portion, wherein the complement portion is substantially complementary to all or a portion of the immobilised primer), a deoxynucleotide, a deoxyribonucleotide, a hydrogen atom instead of a 3’-OH group, a phosphate group, a phosphorothioate group, a propyl spacer (e.g.
  • a modification blocking the 3’-hydroxyl group e.g. hydroxyl protecting groups, such as silyl ether groups (e.g. trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyl(dimethyl)silyl, t-butyl(diphenyl)silyl), ether groups (e.g. benzyl, allyl, t-butyl, methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM), tetrahydropyranyl), or acyl groups (e.g. acetyl, benzoyl)), or an inverted nucleobase.
  • the blocking group may be any modification that prevents extension (i.e. elongation) of the primer by a polymerase.
  • sequence of the sequencing primers and the sequence primer binding sites are not material to the methods of the invention, as long as the sequencing primers are able to bind to the sequence primer binding site to enable amplification and sequencing of the regions to be identified.
  • the first sequencing primer binding site may be selected from ME’- A14’ (as defined in SEQ ID NO. 17 or a variant or fragment thereof), A14’ (as defined in SEQ ID NO. 18 or a variant or fragment thereof), ME’-B15’ (as defined in SEQ ID NO. 19 or a variant or fragment thereof) and B15’ (as defined in SEQ ID NO. 20 or a variant or fragment thereof); and the second sequencing primer binding site may be selected from ME’-HYB2 (as defined in SEQ ID NO. 21 or a variant or fragment thereof), HYB2 (as defined in SEQ ID NO. 11 or a variant or fragment thereof), ME’-HYB2’ (as defined in SEQ ID NO. 22 or a variant or fragment thereof) and HYB2’ (as defined in SEQ ID NO. 13 or a variant or fragment thereof).
  • the first sequencing primer binding site is ME’-B15’ (as defined in SEQ ID NO. 19 or a variant or fragment thereof), and the second sequencing primer binding site is ME’-HYB2’ (as defined in SEQ ID NO. 22 or a variant or fragment thereof).
  • the first sequencing primer binding site is B15’ (as defined in SEQ ID NO.
  • the first and second sequencing primer sites may be located after (e.g. immediately after) a 3’-end of the first and second portions to be identified.
  • the first sequencing primer binding site is ME’-A14’ (as defined in SEQ ID NO. 17 or a variant or fragment thereof), and the second sequencing primer binding site is ME’-HYB2 (as defined in SEQ ID NO. 21 or a variant or fragment thereof).
  • the first sequencing primer binding site may be A14’ (as defined in SEQ ID NO. 18 or a variant or fragment thereof) and the second sequencing primer binding site may be HYB2 (as defined in SEQ ID NO. 11 or a variant or fragment thereof).
  • the first and second sequencing primer sites may be located after (e.g. immediately after) a 3’- end of the first and second portions to be identified.
  • the sequencing primer (which may be referred to herein as the second sequencing primer) comprises or consists of a sequence as defined in SEQ ID NO. 11 to 16, or a variant or fragment thereof.
  • the sequencing primer may further comprise a 3’ blocking group as described above to create a blocked sequencing primer.
  • the primer comprises a 3’-OH group. Such a primer is unblocked and can be elongated with a polymerase.
  • the unblocked and blocked second sequencing primers are present in the sequencing composition in equal concentrations. That is, the ratio of blocked:unblocked second sequencing primers is around 50:50.
  • the sequencing composition may further comprise at least one additional (first) sequencing primer. This additional sequencing primer may be selected from A14-ME (as defined in SEQ ID NO. 9 or a variant or fragment thereof), A14 (as defined in SEQ ID NO. 7 or a variant or fragment thereof), B15-ME (as defined in SEQ ID NO. 10 or a variant or fragment thereof) and B15 (as defined in SEQ ID NO. 8 or a variant or fragment thereof).
  • the sequencing composition comprises blocked second sequencing primers, unblocked second sequencing primers and at least one first sequencing primer, wherein the first sequencing primer is A14, or B15, or is both A14 and B15.
  • selective sequencing may be conducted on the amplified (monoclonal) cluster shown in Figure 6F.
  • a plurality of first sequencing primers 501 are added. These first sequencing primers 501 (e.g. B15-ME; or if ME is not present, then B15) anneal to the first terminal sequencing primer binding site 303 (which represents a type of “first sequencing primer binding site”) (e.g. ME’-B15’; or if ME’ is not present, then B15’).
  • a plurality of second unblocked sequencing primers 502a and a plurality of second blocked sequencing primers 502b are added, either at the same time as the first sequencing primers 501 , or sequentially (e.g. prior to or after addition of first sequencing primers 501).
  • second unblocked sequencing primers 502a e.g. HYB2-ME; or if ME is not present, then HYB2
  • second blocked sequencing primers 502b e.g. blocked HYB2-ME; or if ME is not present, then blocked HYB2
  • an internal sequencing primer binding site in the hybridisation sequence 403’ which represents a type of “second sequencing primer binding site” (e.g. ME’-HYB2’; or if ME’ is not present, then HYB2’).
  • This then allows the first insert complement sequences 40T (i.e. “first portions”) to be sequenced and the second insert complement sequences 402’ (i.e. “second portions”) to be sequenced, wherein a greater proportion of first insert complement sequences 40T are sequenced (grey arrow) compared to a proportion of second insert complement sequences 402’ (black arrow).
  • Figure 8 shows selective sequencing being conducted on a template strand attached to first immobilised primer 201
  • the (monoclonal) cluster may instead have template strands attached to second immobilised primer 202.
  • the first sequencing primers may instead correspond to A14-ME (or if ME is not present, then A14)
  • the second unblocked sequencing primers may instead correspond to HYB2’-ME (or if ME is not present, then HYB2’)
  • second blocked sequencing primers may instead correspond to blocked HYB2’-ME (or if ME is not present, then blocked HYB2’).
  • the positioning of first sequencing primers and second sequencing primers may be swapped. In other words, the first sequencing binding primers may anneal instead to the internal sequencing primer binding site, and the second sequencing binding primers may anneal instead to the terminal sequencing primer binding site.
  • Figure 8 shows concurrent sequencing of a concatenated strand according to the above method.
  • a polynucleotide strand with a first portion (insert) and second portion (insert) can be accurately and simultaneously sequenced by a selective sequencing method that uses a mixture of unblocked and blocked sequencing primers as described above.
  • Figure 9 is a scatter plot showing an example of sixteen distributions of signals generated by polynucleotide sequences disclosed herein.
  • 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.
  • the brighter signal may be A, T, C or G
  • the dimmer signal may be A, T, C or G
  • 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 T and the added nucleobase at the second portion as C.
  • the processor base calls both the added nucleobase at the first portion and the added nucleobase at the second portion as T.
  • the processor base calls the added nucleobase at the first portion as T and the added nucleobase at the second portion as G.
  • the processor base calls the added nucleobase at the first portion as T and the added nucleobase at the second portion as A.
  • the processor base calls the added nucleobase at the first portion as G and the added nucleobase at the second portion as C.
  • the processor base calls the added nucleobase at the first portion as G and the added nucleobase at the second portion as T.
  • the processor base calls both the added nucleobase at the first portion and the added nucleobase at the second portion as G.
  • the processor base calls the added nucleobase at the first portion as G 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.
  • intensity data is obtained.
  • the intensity data includes first intensity data and second intensity data.
  • the first intensity data comprises a combined intensity of a first signal component obtained based upon a respective first nucleobase of the first portion and a second signal component obtained based upon a respective second nucleobase of the second portion.
  • the second intensity data comprises a combined intensity of a third signal component obtained based upon the respective first nucleobase of the first portion and a fourth signal component obtained based upon the respective second nucleobase of the second portion.
  • the first portion is capable of generating a first signal comprising a first signal component and a third signal component.
  • the second portion is capable of generating a second signal comprising a second signal component and a fourth signal component.
  • the first portion and the second portion may be arranged on the solid support such that signals from the first portion and the second portion are detected by a single sensing portion and/or may comprise a single cluster such that first signals and second signals from each of the respective first portions and second portions cannot be spatially resolved.
  • obtaining the intensity data comprises selecting intensity data that corresponds to two (or more) different portions (e.g. the first portion and the second portion).
  • intensity data is selected based upon a chastity score.
  • a chastity score may be calculated as the ratio of the brightest base intensity divided by the sum of the brightest and second brightest base intensities.
  • the desired chastity score may be different depending upon the expected intensity ratio of the light emissions associated with the different portions. As described above, it may be desired to produce clusters comprising the first portion and the second portion, which give rise to signals in a ratio of 2:1.
  • high-quality data corresponding to two portions with an intensity ratio of 2:1 may have a chastity score of around 0.8 to 0.9.
  • the method may proceed to block 1720.
  • one of a plurality of classifications is selected based on the intensity data.
  • Each classification represents a possible combination of respective first and second nucleobases.
  • the plurality of classifications comprises sixteen classifications as shown in Figure 9, each representing a unique combination of first and second nucleobases. Where there are two portions, there are sixteen possible combinations of first and second nucleobases.
  • Selecting the classification based on the first and second intensity data comprises selecting the classification based on the combined intensity of the first and second signal components and the combined intensity of the third and fourth signal components.
  • the method may then proceed to block 1730, where the respective first and second nucleobases are base called based on the classification selected in block 1720.
  • the signals generated during a cycle of a sequencing are indicative of the identity of the nucleobase(s) added during sequencing (e.g. using sequencing-by-synthesis). It will be appreciated that there is a direct correspondence between the identity of the nucleobases that are incorporated and the identity of the complementary base at the corresponding position of the template sequence bound to the solid support. Therefore, any references herein to the base calling of respective nucleobases at the two portions encompasses the base calling of nucleobases hybridised to the template sequences and, alternatively or additionally, the identification of the corresponding nucleobases of the template sequences. The method may then end at block 1740.
  • nucleobases at any given position there are sixteen possible combinations of nucleobases at any given position (i.e. , an A in the first portion and an A in the second portion, an A in the first portion and a T in the second portion, and so on).
  • the light emissions associated with each target sequence during the relevant base calling cycle will be characteristic of the same nucleobase.
  • the two portions behave as a single portion, and the identity of the bases at that position are uniquely callable.
  • the signals associated with each portion in the relevant base calling cycle will be characteristic of different nucleobases.
  • the first signal coming from the first portion have substantially the same intensity as the second signal coming from the second portion.
  • the two signals may also be co-localised, and may not be spatially and/or optically resolved. Therefore, when different nucleobases are present at corresponding positions of the two portions, the identity of the nucleobases cannot be uniquely called from the combined signal alone. However, useful sequencing information can still be determined from these signals.
  • the scatter plot of Figure 11 shows nine distributions (or bins) of intensity values from the combination of two co-localised signals of substantially equal intensity.
  • the intensity values shown in Figure 11 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 first signal generated from the first portion and the second signal generated from the second portion results in a combined signal.
  • the combined signal may be captured by a first optical channel and a second optical channel.
  • the computer system can map the combined signal generated into one of the nine bins, and thus determine sequence information relating to the added nucleobase at the first portion and the added nucleobase at the second portion.
  • Bins are selected based upon the combined intensity of the signals originating from each target sequence during the base calling cycle. For example, bin 1803 may be selected following the detection of a high-intensity (or “on/on”) signal in the first channel and a high-intensity signal in the second channel. Bin 1806 may be selected following the detection of a high-intensity signal in the first channel and an intermediate-intensity (“on/off” or “off/on”) signal in the second channel. Bin 1809 may be selected following the detection of a high-intensity signal in the first channel and a low-intensity or zero- intensity (“off/off”) signal in the second channel.
  • bin 1803 may be selected following the detection of a high-intensity (or “on/on”) signal in the first channel and a high-intensity signal in the second channel.
  • Bin 1806 may be selected following the detection of a high-intensity signal in the first channel and an intermediate-intensity (“on/off” or
  • Bin 1802 may be selected following the detection of an intermediate-intensity signal in the first channel and a high-intensity signal in the second channel.
  • Bin 1805 may be selected following the detection of an intermediate-intensity signal in the first channel and an intermediate-intensity signal in the second channel.
  • Bin 1808 may be selected following the detection of an intermediate- intensity signal in the first channel and a low-intensity or zero-intensity signal in the second channel.
  • Bin 1801 may be selected following the detection of a low-intensity signal in the first channel and a high-intensity signal in the second channel.
  • Bin 1804 may be selected following the detection of a low-intensity or zero-intensity signal in the first channel and an intermediate-intensity signal in the second channel.
  • Bin 1807 may be selected following the detection of a low-intensity or zero-intensity signal in the first channel and a low-intensity signal in the second channel.
  • the computer processor may detect a match between the first portion and the second portion at the sensed position.
  • the computer processor may base call the respective nucleobases. For example, when the combined signal is mapped to bin 1801 for a base calling cycle, the computer processor base calls both the added nucleobase at the first portion and the added nucleobase at the second portion as T.
  • the processor base calls both the added nucleobase at the first portion and the added nucleobase at the second portion as A.
  • the processor base calls both the added nucleobase at the first portion 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 C.
  • bins 1802, 1804, 1806, and 1808 each represent two possible combinations of first and second nucleobases.
  • Bin 1805 meanwhile, represents four possible combinations.
  • mapping the combined signal to an ambiguous bin may still allow for sequencing information to be determined.
  • bins 1802, 1804, 1805, 1806, and 1808 represent mismatches between respective nucleobases of the two portions sensed during the cycle. Therefore, in response to mapping the combined signal to a bin representing a mismatch, the computer processor may detect a mismatch between the first portion and the second portion at the sensed position.
  • bins 1802, 1804, 1806, and 1808 each represent two possible combinations of first and second nucleobases.
  • Bin 1805 meanwhile, represents four possible combinations.
  • mapping the combined signal to an ambiguous bin may still allow for sequencing information to be determined.
  • bins 1802, 1804, 1805, 1806, and 1808 represent mismatches between respective nucleobases of the two portions sensed during the cycle. Therefore, in response to mapping the combined signal to a bin representing a mismatch, the computer processor may detect a mismatch between the first portion and the second portion at the sensed position.
  • A is configured to emit a signal in both the first channel and the second channel
  • C is configured to emit a signal in the first channel only
  • T is configured to emit a signal in the second channel only
  • G does not emit a signal in either channel.
  • different permutations of nucleobases can be used to achieve the same effect by performing dye swaps.
  • A may be configured to emit a signal in both the first channel and the second channel
  • T may be configured to emit a signal in the first channel only
  • C may be configured to emit a signal in the second channel only
  • G may be configured to not emit a signal in either channel.
  • the number of classifications which may be selected based upon the combined signal intensities may be predetermined, for example based on the number of portions expected to be present in the nucleic acid cluster. Whilst Figure 11 shows a set of nine possible classifications, the number of classifications may be greater or smaller.
  • the mapping of the combined signal to each of the different bins can provide additional information about the first portion and the second portion, or about sequences from which the first portion and the second portion were derived. For example, given the nucleic acid material input and the processing methods used to generate the nucleic acid clusters, the first portion and the second portion may be expected to be identical at a given position. In this case, the mapping of the combined signal to a bin representing a mismatch may be indicative of an error introduced during library preparation. In addition, the first portion and the second portion may be expected to be different, for example due to deliberate sequence modifications introduced during library preparation to detect modified cytosines.
  • the library preparation may involve treatment with a conversion agent.
  • the conversion reagent is configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil
  • the correspondence between bases in the original polynucleotide and in the converted strands is shown in Figure 12, alongside a scatter plot showing potential resulting distributions for the combined signal intensities resulting from the simultaneous sequencing of the target sequences.
  • An A-T or T-A base pair in the original molecule will result in a match (A/A or T/T) at the corresponding position of the forward and reverse complement strands of the library.
  • An mC-G or G-mC base pair in the library will also result in a match (G/G or C/C) at the corresponding position of the forward and reverse complement strands of the library.
  • G/G or C/C the conversion of unmodified cytosine to uracil (or a nucleobase which is read as thymine/uracil) in the forward strand of the library (“top” strand)
  • T the corresponding position on the reverse complement strand of the library (“bottom” strand) will be occupied by C.
  • the conversion of unmodified cytosine to uracil (or a nucleobase which is read as thymine/uracil) in the reverse strand of the library (“bottom” strand) will result in an A at the corresponding position of the reverse complement strand of the library. Meanwhile, the corresponding position of the forward strand of the library (“top” strand) will be occupied by G. Therefore, in response to mapping the combined signal to the distribution representing G/G or C/C, the presence of a modified cytosine can be determined at the corresponding position in the original polynucleotide.
  • Figure 13 shows the correspondence between bases in the original polynucleotide and in the converted strands, alongside a scatter plot showing potential resulting distributions for the combined signal intensities resulting from the simultaneous sequencing of the target sequences.
  • An A-T or T-A base pair in the library will result in a match (A/A or T/T) at the corresponding position of the forward and reverse complement strands of the library.
  • a C-G or G-C base pair in the library will also result in a match (G/G or C/C) at the corresponding position of the forward and reverse complement strands of the library.
  • the conversion of 5-methylcytosine to thymine in the forward strand of the library (“top” strand) will result in a T at the corresponding position of the forward strand of the library. Meanwhile, the corresponding position on the reverse complement strand of the library (“bottom” strand) will be occupied by C.
  • the conversion of 5-methylcytosine to thymine in the reverse strand of the library (“bottom” strand) will result in an A at the corresponding position of the reverse complement strand of the library. Meanwhile, the corresponding position of the forward strand of the library (“top” strand) will be occupied by G. Therefore, in response to mapping the combined signal to the distribution representing an A/G, G/A, T/C, or C/T mismatch, the presence of a modified cytosine can be determined at the corresponding position in the original polynucleotide.
  • Figure 14 represents the distributions resulting from the use of an alternative dye- encoding scheme following use of a conversion reagent configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil
  • Figure 15 represents the distributions resulting from the use of an alternative dye-encoding scheme following use of a conversion reagent configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil.
  • Figure 16 represents yet another distribution resulting from the use of an alternative dye- encoding scheme following use of a conversion reagent configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil.
  • modified cytosines fall within a central bin.
  • the dye-encoding scheme may be optimised to allow for different combinations of first and second nucleobases to be resolved. This may be particularly useful where sequence modifications of a known type have been introduced into the first portions and the second portions. For example, where sequence modifications have been introduced that result in the conversion of unmodified cytosines to uracil or nucleobases which is read as thymine/uracil, or the conversion of modified cytosines to thymine or nucleobases which are read as thymine/uracil, the dye-encoding scheme may be selected such that the resulting combination of first and second nucleobases do not fall within the central bin (which represents four different nucleobase combinations).
  • a T/C or G/A mismatch between the forward and reverse complement strands is indicative of the presence of a mC-G or G-mC base pair at the corresponding position of the library.
  • the dye-encoding scheme may therefore be designed such that these mismatches may be resolved from other possible combinations of nucleobases. This may be achieved by detecting light emissions from A and T bases in a first illumination cycle, and from C and T bases in a second illumination cycle. In another example, light emissions may be detected from C and G bases in a first illumination cycle, and from C and T bases in a second illumination cycle. In another example, light emissions may be detected from C and A bases in a first illumination cycle, and from C and G bases in a second illumination cycle.
  • a C/C or G/G match between the forward and reverse complement strands is indicative of the presence of a mC-G or G-mC base pair at the corresponding position of the library.
  • a mC-G or G-mC base pair will always be resolvable.
  • the dye-encoding scheme can still be designed to optimise the resolution between unmodified bases.
  • Figure 17 is a flow diagram showing a method 1900 of determining sequence information according to the present disclosure.
  • the described method allows for the determination of sequence information from 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.
  • the disclosed method 1900 may start from block 1901. The method may then move to block 1910.
  • intensity data is obtained.
  • the intensity data includes first intensity data and second intensity data.
  • the first intensity data comprises a combined intensity of a first signal component obtained based upon a respective first nucleobase of the first portion and a second signal component obtained based upon a respective second nucleobase of the second portion.
  • the second intensity data comprises a combined intensity of a third signal component obtained based upon the respective first nucleobase of the first portion and a fourth signal component obtained based upon the respective second nucleobase of the second portion.
  • the first portion is capable of generating a first signal comprising a first signal component and a third signal component.
  • the second portion is capable of generating a second signal comprising a second signal component and a fourth signal component.
  • the first portion and the second portion may be arranged on the solid support such that signals from the first portion and the second portion are detected by a single sensing portion and/or may comprise a single cluster such that first signals and second signals from each of the respective first portions and second portions cannot be spatially resolved.
  • obtaining the intensity data comprises selecting intensity data, for example based upon a chastity score.
  • a chastity score may be calculated as the ratio of the brightest base intensity divided by the sum of the brightest and second brightest base intensities.
  • high-quality data corresponding to two portions with a substantially equal intensity ratio may have a chastity score of around 0.8 to 0.9, for example 0.89-0.9.
  • the method may proceed to block 1920.
  • one of a plurality of classifications is selected based on the intensity data.
  • Each classification represents one or more possible combinations of respective first and second nucleobases, and at least one classification of the plurality of classifications represents more than one possible combination of respective first and second nucleobases.
  • the plurality of classifications comprises nine classifications as shown in Figure 11 . Selecting the classification based on the first and second intensity data comprises selecting the classification based on the combined intensity of the first and second signal components and the combined intensity of the third and fourth signal components.
  • the method may then proceed to block 1930, where sequence information of the respective first and second nucleobases is determined based on the classification selected in block 1920.
  • the signals generated during a cycle of a sequencing are indicative of the identity of the nucleobase(s) added during sequencing (e.g. using sequencing-by-synthesis). For example, it may be determined that there is a match or a mismatch between the respective first and second nucleobases. Where it is determined that there is a match between the first and second respective nucleobases, the nucleobases may be base called. Whether there is a match or a mismatch, additional or alternative information may be obtained, as described above.
  • any references herein to the base calling of respective nucleobases at the two portions encompasses the base calling of nucleobases hybridised to the template sequences and, alternatively or additionally, the identification of the corresponding nucleobases of the template sequences.
  • the method may then end at block 1940.
  • the present invention is directed to methods of preparing a polynucleotide strand for detection of modified cytosines, such that where the strand comprises two portions (in other words, a concatenated polynucleotide sequence comprising a first portion and a second portion) to be identified, the first portion comprising a forward strand and the second portion comprising a reverse complement strand (or the first portion comprising a reverse strand and the second portion comprising a forward complement strand), such portions can be identified concurrently, thus facilitating the detection of modified cytosines.
  • the methods of the present invention allow a decrease in the amount of time taken to detect modified cytosines.
  • selective processing methods may be used when preparing the templates. This leads to further advantages, as it also becomes possible to identify which strand of the original library that the modified cytosine was on, whilst maintaining reductions in time taken to detect modified cytosines.
  • a method of preparing at least one polynucleotide sequence for detection of modified cytosines comprising: synthesising at least one polynucleotide sequence comprising a first portion and a second portion, wherein the at least one polynucleotide sequence comprises portions of a double-stranded nucleic acid template, and the first portion comprises a forward strand of the template, and the second portion comprises a reverse complement strand of the template; or wherein the first portion comprises a reverse strand of the template, and the second portion comprises a forward complement strand of the template, wherein the template is generated from a (double-stranded) target polynucleotide to be sequenced via complementary base pairing, and wherein the target polynucleotide has been pre-treated using a conversion reagent, wherein the conversion reagent is configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil, and/or where
  • the at least one polynucleotide sequence comprises portions of a double-stranded nucleic acid template
  • the first portion may comprise (or be) the forward strand of a polynucleotide sequence (e.g. forward strand of a template)
  • the second portion may comprise (or be) the reverse complement strand of the polynucleotide sequence (e.g. reverse complement strand of the template) (in effect, a reverse complement strand may be considered a “copy” of the forward strand).
  • the first portion may comprise (or be) the reverse strand of a polynucleotide sequence (e.g.
  • the second portion may comprise (or be) the forward complement strand of the polynucleotide sequence (e.g. forward complement strand of the template) (in effect, a forward complement may be considered a “copy” of the reverse strand).
  • the first portion may be derived from a forward strand of a target polynucleotide to be sequenced, and the second portion may be derived from a reverse complement strand of the target polynucleotide to be sequenced; or the first portion may be derived from a reverse strand of a target polynucleotide to be sequenced, and the second portion may be derived from a forward complement strand of the target polynucleotide to be sequenced.
  • the template is generated from a (double-stranded) target polynucleotide to be sequenced via complementary base pairing.
  • the (double-stranded) target polynucleotide may be one (double-stranded) polynucleotide present in a polynucleotide library to be sequenced.
  • the template allows sequence information to be obtained for that particular polynucleotide.
  • the method may further comprise a step of preparing the first portion and the second portion for concurrent sequencing.
  • the method may comprise simultaneously contacting first sequencing primer binding sites located after a 3’-end of the first portions with first primers and second sequencing primer binding sites located after a 3’-end of the second portions with second primers.
  • first portions and second portions are primed for concurrent sequencing.
  • a proportion of first portions may be capable of generating a first signal and a proportion of second portions may be capable of generating a second signal, wherein an intensity of the first signal is substantially the same as an intensity of the second signal.
  • a proportion of first portions may be capable of generating a first signal and a proportion of second portions may be capable of generating a second signal, wherein an intensity of the first signal is substantially the same as an intensity of the second signal.
  • the first signal and the second signal may be spatially unresolved (e.g. generated from the same region or substantially overlapping regions).
  • the first portion may be referred to herein as read 1 (R1).
  • the second portion may be referred to herein as read 2 (R2).
  • the first portion is at least 25 or at least 50 base pairs and the second portion is at least 25 base pairs or at least 50 base pairs.
  • the single (concatenated) polynucleotide strand may be attached to a solid support.
  • this solid support is a flow cell.
  • the polynucleotide strand is attached to the solid support in a single well of the solid support.
  • the polynucleotide strand or strands may form or be part of a cluster on the solid support.
  • cluster may refer to a clonal group of template polynucleotides (e.g. DNA or RNA) bound within a single well of a solid support (e.g. flow cell).
  • a cluster may refer to the population of polynucleotide molecules within a well that are then sequenced.
  • a “cluster” may contain a sufficient number of copies of template polynucleotides such that the cluster is able to output a signal (e.g. a light signal) that allows sequencing reads to be performed on the cluster.
  • a “cluster” may comprise, for example, about 500 to about 2000 copies, about 600 to about 1800 copies, about 700 to about 1600 copies, about 800 to 1400 copies, about 900 to 1200 copies, or about 1000 copies of template polynucleotides.
  • a cluster may be formed by bridge amplification, as described above.
  • one group of strands may be removed from the solid support, leaving either the templates or the template complements, as explained above.
  • a cluster may be considered to be a “monoclonal” cluster.
  • a “monoclonal” cluster is meant that the population of polynucleotide sequences that are then sequenced (as the next step) are substantially the same - i.e. copies of the same sequence.
  • a “monoclonal” cluster may refer to the population of single polynucleotide molecules within a well that are then sequenced.
  • a “monoclonal” cluster may contain a sufficient number of copies of a single template polynucleotide (or copies of a single template complement polynucleotide) such that the cluster is able to output a signal (e.g. a light signal) that allows sequencing reads to be performed on the “monoclonal” cluster.
  • a signal e.g. a light signal
  • a “monoclonal” cluster may comprise, for example, about 500 to about 2000 copies, about 600 to about 1800 copies, about 700 to about 1600 copies, about 800 to 1400 copies, about 900 to 1200 copies, or about 1000 copies of a single template polynucleotide (or copies of a single template complement polynucleotide).
  • the copies of the single template polynucleotide (and/or single template complement polynucleotides) may comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 95%, 98%, 99% or 100% of all polynucleotides within a single well of the flow cell, and thus providing a substantially monoclonal “cluster”.
  • the at least one polynucleotide sequence comprising a first portion and a second portion may be prepared using a tandem insert method as described herein. Accordingly, in one embodiment, the step of synthesising the at least one polynucleotide sequence comprising a first portion and a second portion may comprise: synthesising a first precursor polynucleotide fragment comprising a complement of the first portion and a hybridisation complement sequence, synthesising a second precursor polynucleotide fragment comprising a second portion and a hybridisation sequence, annealing the hybridisation complement sequence of the first precursor polynucleotide fragment with the hybridisation sequence on the second precursor polynucleotide fragment to form a hybridised adduct, synthesising a first precursor polynucleotide sequence by extending the first precursor polynucleotide fragment to form a complement of the second portion, and synthesising the at least one polynucleotide sequence by forming a complement of the first precursor polynucle
  • the first precursor polynucleotide fragment may comprise a first sequencing primer binding site complement.
  • the first sequencing primer binding site complement may be located before a 5’-end of the complement of the first portion, such as immediately before the 5’- end of the complement of the first portion.
  • the first precursor polynucleotide fragment may comprise a second adaptor complement sequence.
  • the second adaptor complement sequence may be located before a 5’-end of the complement of the first portion.
  • the first precursor polynucleotide fragment may comprise a first sequencing primer binding site complement and a second adaptor complement sequence.
  • the first sequencing primer binding site complement may be located before a 5’-end of the complement of the first portion, and wherein the second adaptor complement sequence may be located before a 5’-end of the first sequencing primer binding site complement.
  • the first precursor polynucleotide fragment may comprise a second sequencing primer binding site complement.
  • the hybridisation sequence complement may comprise the second sequencing primer binding site complement.
  • the second precursor polynucleotide fragment may comprise a first adaptor complement sequence.
  • the method may further comprise a step of concurrently sequencing nucleobases in the first portion and the second portion.
  • the target polynucleotide (or in some embodiments, the polynucleotide library) has been pre-treated using a conversion reagent.
  • the method of preparing at least one polynucleotide sequence for detection of modified cytosines may include a step of treating the target polynucleotide using a conversion agent.
  • Figure 18 shows the effect of the pre-treatment of the target polynucleotide of various conversion agents on the bases in the resulting template strands.
  • the conversion reagent is configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil, and/or is configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil.
  • modified cytosine may refer to any one or more of 5- methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and
  • unmodified cytosine refers to cytosine (C): cytosine
  • the term “conversion reagent configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil” may refer to a reagent which converts one or more modified cytosines (e.g. 5-methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine) to thymine (i.e. would base pair with adenine), or to an equivalent nucleobase which would base pair with adenine.
  • the conversion may comprise a deamination reaction converting the modified cytosine to thymine or nucleobase which is read as thymine/uracil.
  • the term “conversion reagent configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil” may refer to a reagent which converts one or more unmodified cytosines to uracil (i.e. would base pair with adenine), or to an equivalent nucleobase which would base pair with adenine.
  • the conversion may comprise a deamination reaction converting the unmodified cytosine to uracil or nucleobase which is read as thymine/uracil.
  • the forward strand of the template will then not be identical to the reverse complement strand of the template as a result of treatment of the target polynucleotide with the conversion agent (alternatively, the reverse strand of the template will then not be identical to the forward complement strand of the template as a result of treatment of the target polynucleotide with the conversion agent).
  • the forward strand of the template will then be (substantially) identical to the reverse complement strand of the template despite treatment of the target polynucleotide with the conversion agent (alternatively, the reverse strand of the template will then be (substantially) identical to the forward complement strand of the template despite treatment of the target polynucleotide with the conversion agent).
  • mismatches between the forward strand of the template and the reverse complement strand of the template allow the detection of modified cytosines (alternatively, mismatches between the reverse strand of the template and the forward complement strand of the template allow detection of modified cytosines).
  • the forward strand (or reverse strand) of the template may comprise a guanine base at a first position, which leads to a basecall of C for the original target polynucleotide; and wherein the reverse complement strand (or forward complement strand) of the template may comprise an adenine base at a second position corresponding to the same position number as the first position, which leads to a basecall of T for the original target polynucleotide.
  • the adenine base at the second position within the template may have been generated as a result of conversion of modified cytosines in the target polynucleotide to thymine, or to an equivalent nucleobase which would base pair with adenine; or may have been generated as a result of conversion of unmodified cytosines in the target polynucleotide to uracil, or to an equivalent nucleobase which would base pair with adenine.
  • the adenine base at the second position within the template may have been generated as a result of conversion of unmodified cytosines in the target polynucleotide to uracil, or to an equivalent nucleobase which would base pair with adenine.
  • the forward strand (or reverse strand) of the template comprises an adenine base at a first position, which leads to a basecall of T for the original target polynucleotide; and wherein the reverse complement strand (or forward complement strand) of the template comprises a guanine base at a second position corresponding to the same position number as the first position, which leads to a basecall of C for the original target polynucleotide.
  • the adenine base at the first position within the template may have been generated as a result of conversion of modified cytosines in the target polynucleotide to thymine, or to an equivalent nucleobase which would base pair with adenine; or may have been generated as a result of conversion of unmodified cytosines in the target polynucleotide to uracil, or to an equivalent nucleobase which would base pair with adenine.
  • the adenine base at the first position within the template may have been generated as a result of conversion of modified cytosines in the target polynucleotide to thymine, or to an equivalent nucleobase which would base pair with adenine.
  • the conversion reagent configured to convert s modified cytosine to thymine or a nucleobase which is read as thymine/uracil may further be configured to be selective for converting one or more modified cytosines (e.g. 5-methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine) over converting unmodified cytosine.
  • modified cytosines e.g. 5-methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine
  • the selectivity may be measured by comparing reaction parameters (e.g. deamination reaction parameters) of the conversion of a particular modified cytosine to thymine or equivalent nucleobase which is read as thymine/uracil, with corresponding reaction parameters (e.g.
  • deamination reaction parameters of the conversion of unmodified cytosine to uracil or nucleobase which is read as thymine/uracil.
  • reaction parameters such as rate of reaction or yield may be compared.
  • rate of reaction a rate of a reaction (e.g. deamination) of the particular modified cytosine to thymine or nucleobase which is read as thymine/uracil may be greater (e.g. at least 2 times greater, at least 5 times greater, at least 10 times greater, at least 20 times greater, at least 50 times greater, or at least 100 times greater) than a corresponding rate of a reaction (e.g.
  • a yield of a reaction (e.g. deamination) of the particular modified cytosine to thymine or nucleobase which is read as thymine/uracil may be greater (e.g. at least 2 times greater, at least 5 times greater, at least 10 times greater, at least 20 times greater, at least 50 times greater, or at least 100 times greater) than a corresponding yield of a reaction (e.g. deamination) of the unmodified cytosine to uracil or nucleobase which is read as thymine/uracil.
  • the conversion reagent configured to convert an unmodified cytosine to uracil or a nucleobase which is read as thymine/uracil may further be configured to be selective for converting unmodified cytosine over converting one or more modified cytosines (e.g. 5-methylcytosine, 5-hydroxymethylcytosine, 5- formylcytosine and 5-carboxylcytosine).
  • modified cytosines e.g. 5-methylcytosine, 5-hydroxymethylcytosine, 5- formylcytosine and 5-carboxylcytosine.
  • the selectivity may be measured by comparing reaction parameters (e.g. deamination reaction parameters) of the conversion of unmodified cytosine to uracil or nucleobase which is read as thymine/uracil, with corresponding reaction parameters (e.g.
  • deamination reaction parameters of the conversion of a particular modified cytosine to thymine or nucleobase which is read as thymine/uracil.
  • reaction parameters such as rate of reaction or yield may be compared.
  • rate of reaction a rate of a reaction (e.g. deamination) of the unmodified cytosine to uracil or nucleobase which is read as thymine/uracil may be greater (e.g. at least 2 times greater, at least 5 times greater, at least 10 times greater, at least 20 times greater, at least 50 times greater, or at least 100 times greater) than a rate of a reaction (e.g.
  • a yield of a reaction (e.g. deamination) the unmodified cytosine to uracil or nucleobase which is read as thymine/uracil may be greater (e.g. at least 2 times greater, at least 5 times greater, at least 10 times greater, at least 20 times greater, at least 50 times greater, or at least 100 times greater) than a corresponding yield of a reaction (e.g. deamination) of the particular modified cytosine to uracil or the nucleobase which is read as thymine/uracil.
  • the conversion agent may comprise a chemical agent and/or an enzyme.
  • the chemical agent may comprise a boron-based reducing agent.
  • the boron-based reducing agent is an amine-borane compound or an azine-borane compound (wherein the term “azine” refers to a nitrogenous heterocyclic compound comprising a 6-membered aromatic ring).
  • amine- borane compounds include compounds such as t-butylamine borane, ammonia borane, ethylenediamine borane and dimethylamine borane.
  • Non-limiting examples of azine- borane compounds include compounds such as pyridine borane and 2-picoline borane.
  • boron-based reducing agents are able to convert 5-formylcytosine and 5- carboxylcytosine to dihydrouracil (i.e. a nucleobase which is read as thymine/uracil).
  • This process is selective for a particular type of modified cytosine (5-carboxylcytosine) and does not convert unmodified cytosine.
  • modified cytosine 5-carboxylcytosine
  • treatment with further agents as described herein prior to treatment with the boron-based reducing agent may provide such distinction.
  • boron- based reducing agents may be combined with ten-eleven translocation (TET) methylcytosine dioxygenases, p-glucosyltransferases, oxidising agents, oximes and/or hydrazones as described herein.
  • TET ten-eleven translocation
  • the chemical agent may comprise sulfite.
  • the sulfite may be present in a partially acid/salt form (e.g. as bisulfite ions), or be present in a salt form (e.g. as sulfite ions).
  • the sulfite may comprise a cation (not including H + ).
  • the cation may be selected from “metal cations” or “non-metal cations”.
  • Metal cations may include alkali metal ions (e.g. lithium, sodium, potassium, rubidium or caesium ions).
  • Non-metal cations may include ammonium salts (e.g.
  • alkylammonium salts or phosphonium salts (e.g. alkylphosphonium salts).
  • phosphonium salts e.g. alkylphosphonium salts.
  • sulfite also encompasses “metabisulfite”, which dissolves in aqueous solution to form bisulfite.
  • sulfite e.g. bisulfite
  • sulfite is able to convert unmodified cytosine to uracil.
  • modified cytosine 5-methylcytosine and 5-hydroxymethylcytosine
  • 5-formylcytosine and 5- carboxylcytosine are converted to their equivalent deaminated versions.
  • treatment with further agents as described herein prior to treatment with the sulfite may provide such distinction.
  • the sulfite may be combined with ten-eleven translocation (TET) methylcytosine dioxygenases, p- glucosyltransferases, oxidising agents and/or reducing agents as described herein.
  • the enzyme may comprise a cytidine deaminase.
  • cytidine deaminase may refer to an enzyme which is able to catalyse the following reaction: wherein R is hydrogen, methyl, hydroxymethyl, formyl or carboxyl, and wherein the wavy line indicates an attachment point to a polynucleotide.
  • the cytidine deaminase is a wild-type cytidine deaminase or a mutant cytidine deaminase. In one example, the cytidine deaminase is a mutant cytidine deaminase.
  • the cytidine deaminase is a member of the APOBEC protein family. In one embodiment, the cytidine deaminase is a member of the AID subfamily, the APOBEC1 subfamily, the APOBEC2 subfamily, the APOBEC3 subfamily (e.g.
  • the cytidine deaminase is a member of the APOBEC3A subfamily.
  • cytidine deaminases are able to catalyse the deamination of all modified cytosines (particularly 5-methylcytosine, 5-hydroxymethylcytosine and 5-formylcytosine) to their equivalent deaminated versions (i.e. nucleobases which are read as thymine/uracil), as well as catalysing the deamination of unmodified cytosines to uracil.
  • rates of reaction may differ depending on the type of modified cytosine; for example, wild-type APOBEC3A catalyses the deamination of unmodified cytosine and 5-methylcytosine relatively efficiently, whereas deamination of 5- hydroxymethylcytosine is ⁇ 5000-fold slower relative to unmodified cytosine, deamination of 5-formylcytosine is ⁇ 3700-fold slower relative to unmodified cytosine, and deamination of 5-carboxylcytosine is >20000-fold slower relative to unmodified cytosine.
  • modified cytosines and unmodified cytosines are desired (or even between different types of modified cytosines)
  • treatment with further agents as described herein prior to treatment with the cytidine deaminase may provide such distinction.
  • the cytidine deaminase may be combined with ten-eleven translocation (TET) methylcytosine dioxygenases and/or p-glucosyltransferases as described herein.
  • TET ten-eleven translocation
  • particular cytidine deaminases e.g. mutant cytidine deaminases
  • the APOBEC protein family is a member of the large cytidine deaminase superfamily that contains a canonical zinc-dependent deaminase (ZDD) signature motif embedded within a core cytidine deaminase fold.
  • This fold includes a five-stranded mixed beta (b)- sheet surrounded by six alpha (a)-helices with the order a1-b1-b2-a2-b3-a3-b4-a4-b5- a5-a6 (Salter et al., Trends Biochem Sci. 2016 41 (7):578-594. doi: 10.1016/j.tibs.2016.05.001 ; Salter et al., Trends Biochem. Sci.
  • Each cytidine deaminase domain core structure of APOBEC proteins contains a highly conserved spatial arrangement of the catalytic centre residues of a zinc-binding motif H-[P/A/V]-E-X[23-28]-P-C-X[2-4]-C (SEQ ID NO. 67) (referred to herein as the ZDD motif, where X is any amino acid, and the subscript range of numbers after X refers to the number of amino acids) (Salter et al., Trends Biochem Sci. 201641(7):578-594.
  • Some members of the APOBEC protein family include one copy of the ZDD motif.
  • Other members of the APOBEC protein family e.g., the APOBEC3B subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, and the APOBEC3G subfamily, include two copies of the ZDD motif, but often only the C-terminal copy is active (Salter et al., Trends Biochem Sci.
  • a mutant cytidine deaminase disclosed herein includes one or two ZDD motifs.
  • a mutant cytidine deaminase based on a member of the APOBEC3A subfamily includes the following ZDD motif: HXEX24SW(S/T)PCX [ 2-4]CX6FX8LX5R(L/l)YX [ 8-ii]LX 2 LX [ io]M (SEQ ID NO.
  • Non-limiting examples of wild-type cytidine deaminases in the APOBEC protein family are shown in the table below (from UniProt, database of protein sequence and functional information, available at uniprot.org; or GenBank, collection of nucleotide sequences and their protein translations, available at ncbi.nlm.nih.gov/protein/):
  • the mutant cytidine deaminase may comprise amino acid substitution mutations at positions functionally equivalent to (Tyr/Phe)130 and Tyr132 in a wild-type APOBEC3A protein.
  • Such mutant cytidine deaminases are described in further detail in US Provisional Application 63/328,444, which is incorporated herein by reference.
  • “functionally equivalent” it is meant that the mutant cytidine deaminase has the amino acid substitution at the amino acid position in a reference (wild-type) cytidine deaminase that has the same functional role in both the reference (wild-type) cytidine deaminase and the mutant cytidine deaminase.
  • the (Tyr/Phe)130 may be Tyr130, and the wild-type APOBEC3A protein may be SEQ ID NO. 32.
  • the mutant cytidine deaminase may convert 5-methylcytosine to thymine by deamination at a greater rate than conversion rate of cytosine to uracil by deamination; wherein the rate may be at least 100-fold greater.
  • substitution mutation at the position functionally equivalent to Tyr130 may comprise Ala, Vai or Trp.
  • the substitution mutation at the position functionally equivalent to Tyr132 may comprise a mutation to His, Arg, Gin or Lys.
  • the mutant cytidine deaminase may comprise a ZDD motif H- [P/A/V]-E-X [2 3-28]-P-C-X [2 -4]-C (SEQ ID NO. 67).
  • the mutant cytidine deaminase may be a member of the APOBEC3A subfamily and may comprise a ZDD motif HXEX24SW(S/T)PCX[2-4]CX6FX8LX5R(L/I)YX[8- njLXzLXnoiM (SEQ ID NO. 68).
  • the target polynucleotide may be treated with a further agent prior to treatment with the conversion reagent.
  • the further agent may be configured to convert a modified cytosine (e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5- carboxylcytosine) to another modified cytosine (e.g. another one of 5-methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine).
  • a modified cytosine e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5- carboxylcytosine
  • another modified cytosine e.g. another one of 5-methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.
  • the further agent may be configured to convert 5-methylcytosine to 5- hydroxymethylcytosine.
  • the further agent may be configured to convert 5-hydroxymethylcytosine to 5-formylcytosine.
  • the further agent may be configured to convert 5-formylcytosine to 5- carboxylcytosine.
  • the further agent may be configured to convert 5-methylcytosine to 5-hydroxymethylcytosine, 5-hydroxymethylcytosine to 5- formylcytosine, and 5-formylcytosine to 5-carboxylcytosine.
  • the further agent may be configured to convert 5-formylcytosine to 5-hydroxymethylcytosine.
  • the further agent configured to convert a modified cytosine e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5- carboxylcytosine
  • a modified cytosine e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5- carboxylcytosine
  • another modified cytosine e.g. another (different) one of 5- methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine
  • a chemical agent and/or an enzyme e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine
  • the further agent configured to convert a modified cytosine to another modified cytosine may be a chemical agent; for example, an oxidising agent; such as a metal-based oxidising agent; such as a transition metal-based oxidising agent; such as a ruthenium- based oxidising agent.
  • the oxidising agent may be configured to convert 5- hydroxymethylcytosine to 5-formylcytosine.
  • Non-limiting examples of the oxidising agent include ruthenate (e.g. potassium ruthenate, K2RUO4), or perruthenate (e.g. potassium perruthenate, KRUO4).
  • the further agent configured to convert a modified cytosine to another modified cytosine may be a chemical agent; for example, a reducing agent; such as a Group Ill-based reducing agent; for example, a boron-based reducing agent.
  • the oxidising agent may be configured to convert 5-formylcytosine to 5-hydroxymethylcytosine.
  • Non-limiting examples of the reducing agent include borohydride (e.g. sodium borohydride, lithium borohydride), or triacetoxyborohydride (e.g. sodium triacetoxyborohydride).
  • the further agent configured to convert a modified cytosine to another modified cytosine may be an enzyme; such as a ten-eleven translocation (TET) methylcytosine dioxygenase; wherein the TET methylcytosine dioxygenase may be a member of the TET1 subfamily, the TET2 subfamily, or the TET3 subfamily.
  • the enzyme may be configured to convert 5-methylcytosine to 5-hydroxymethylcytosine, 5- hydroxymethylcytosine to 5-formylcytosine, and 5-formylcytosine to 5-carboxylcytosine.
  • TET methylcytosine dioxygenase include:
  • the further agent may be configured to reduce/prevent deamination of a particular modified cytosine (e.g. one of 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine).
  • a further agent configured to reduce/prevent deamination of a particular modified cytosine may be used in combination with a further agent configured to convert a modified cytosine to another modified cytosine.
  • the further agent may be configured to convert 5-hydroxymethylcytosine to a 5-hydroxymethylcytosine analogue bearing a hydroxyl protecting group.
  • the 5- hydroxymethylcytosine analogue bearing the hydroxyl protecting group may be resistant to oxidation to form 5-formylcytosine.
  • hydroxyl protecting groups include sugar groups (e.g. glycosyl), silyl ether groups (e.g. trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyl(dimethyl)silyl, t-butyl(diphenyl)silyl), ether groups (e.g.
  • benzyl allyl, t-butyl, methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM), tetrahydropyranyl), or acyl groups (e.g. acetyl, benzoyl).
  • the further agent may be configured to convert 5-formylcytosine to a 5-formylcytosine analogue bearing an oxime or a hydrazone group.
  • the 5- formylcytosine analogue bearing the oxime or hydrazone group may be resistant to oxidation to form 5-carboxylcytosine.
  • the further agent configured to reduce/prevent deamination of a particular modified cytosine may comprise a chemical agent and/or an enzyme.
  • the further agent configured to reduce/prevent deamination of a particular modified cytosine may be an enzyme; for example, a glycosyltransferase (e.g. a- glucosyltransferase or p-glucosyltransferase); such as a p-glucosyltransferase.
  • a further agent may be configured to convert 5-hydroxymethylcytosine to a 5- hydroxymethylcytosine analogue bearing a hydroxyl protecting group, wherein the hydroxyl protecting group is glycosyl.
  • a non-limiting example of the enzyme includes T4- PGT, for example as supplied by New England BioLabs (catalog # M0357S, M0357L) or by ThermoFisher Scientific (catalog # EO0831); further non-limiting examples of glycosyltransferases include:
  • the further agent configured to reduce/prevent deamination of a particular modified cytosine may be a chemical agent; such as a hydroxylamine or a hydrazine. Such a further agent may be configured to convert 5-formylcytosine to a 5-formylcytosine analogue bearing an oxime or a hydrazone group.
  • hydroxylamines include O-alkylhydroxylamines (e.g. O-methylhydroxylamine, O- ethylhydroxylamine), O-arylhydroxylamines (e.g. O-phenylhydroxylamine).
  • Non-limiting examples of hydrazines include acylhydrazides (e.g.
  • acethydrazide benzhydrazide
  • alkylsulfonylhydrazides e.g. methylsulfonylhydrazide
  • arylsulfonylhydrazides e.g. benzenesulfonylhydrazide, p-toluenesulfonylhydrazide
  • specific methods of modified cytosine sequencing using conversion agents are further illustrated below. However, the type of conversion agents and/or further agents are not limited thereto.
  • Bisulfite sequencing involves using bisulfite as the conversion agent. This process is described in Frommer et al. (Proc. Natl. Acad. Sci. U.S.A., 1992, 89, pp. 1827- 1831), which is incorporated herein by reference. This process converts unmodified cytosines in the target polynucleotide to uracil, as well as 5-formylcytosine and 5- carboxylcytosine to deaminated analogues, but does not convert 5-methylcytosine and 5-hydroxymethylcytosine. Accordingly, BS-seq allows identification of the modified cytosines 5-mC and 5-hmC by reading them as C; whereas unmodified C, 5-fC and 5- caC are converted to nucleobases which are read as T/U.
  • Oxidative bisulfite sequencing involves using potassium perruthenate as the further agent and bisulfite as the conversion agent. This process is described in Booth et al. (Science, 2012, 336, pp. 934-937), which is incorporated herein by reference. Potassium perruthenate causes oxidation of 5-hydroxymethylcytosine in the target polynucleotide to 5-formylcytosine.
  • cytosines in the target polynucleotide to uracil, as well as 5-formylcytosine (including residues that used to be 5-hydroxymethylcytosine) and 5-carboxylcytosine to deaminated analogues, but does not convert 5-methylcytosine.
  • oxBS-seq allows identification of the modified cytosine 5-mC by reading them as C; whereas unmodified C, 5-hmC, 5-fC and 5-caC are converted to nucleobases which are read as T/U.
  • Reduced bisulfite sequencing involves using sodium borohydride as the further agent and bisulfite as the conversion agent. This process is described in Booth et al. (Nat. Chem., 2014, 6, pp. 435-440), which is incorporated herein by reference.
  • Sodium borohydride causes reduction of 5-formylcytosine in the target polynucleotide to 5-hydroxymethylcytosine.
  • redBS-seq allows identification of the modified cytosines 5-mC, 5-hmC and 5-fC by reading them as C; whereas unmodified C and 5-caC are converted to nucleobases which are read as T/ll.
  • TET-assisted bisulfite sequencing involves using a T4 bacteriophage p- glucosyltransferase and a TET1 enzyme as the further agents and bisulfite as the conversion agent. This process is described in Yu et al. (Cell, 2012, 149, pp. 1368-1380), which is incorporated herein by reference.
  • the T4 bacteriophage p-glucosyltransferase converts 5-hydroxymethylcytosine in the target polynucleotide to p-glucosyl-5- hydroxymethylcytosine, which prevents oxidation.
  • TET1 enzyme causes oxidation of 5- methylcytosine and 5-formylcytosine in the target polynucleotide to 5-carboxylcytosine.
  • Subsequent treatment with bisulfite converts unmodified cytosines in the target polynucleotide to uracil, as well as 5-carboxylcytosine (including residues that used to be 5-methylcytosine and 5-formylcytosine) to its deaminated analogue, but does not convert p-glucosyl-5-hydroxymethylcytosine.
  • TAB-seq allows identification of the modified cytosine 5-hmC (as the protected glycosyl residue) by reading it as C; whereas unmodified C, 5-mC, 5-fC and 5-caC are converted to nucleobases which are read as T/ll.
  • APOBEC-coupled epigenetic sequencing involves using a T4 bacteriophage P-glucosyltransferase as a further agent and APOBEC3A as the conversion agent. This process is described in Schutsky et al. (Nat. Biotechnol., 2018, 36, pp. 1083-1090), which is incorporated herein by reference.
  • the T4 bacteriophage p-glucosyltransferase converts 5-hydroxymethylcytosine in the target polynucleotide to p-glucosyl-5- hydroxymethylcytosine, which prevents oxidation.
  • Subsequent treatment with APOBEC3A converts unmodified cytosines in the target polynucleotide to uracil, as well as 5-methylcytosine to its deaminated analogue.
  • 5-formylcytosine is also able to convert to its deaminated analogue, but reacts slower relative to unmodified cytosine and 5- methylcytosine.
  • 5-carboxylcytosine is also able to convert to its deaminated analogue, but reacts far slower than unmodified cytosine and 5-methylcytosine, and slower than 5- formylcytosine.
  • ACE-seq allows identification of the modified cytosine 5- hmC (as the protected glycosyl residue) by reading it as C; whereas unmodified C and 5-mC are converted to nucleobases which are read as T/ll; 5-fC is converted to a nucleobase which is read as T/ll to a limited extent; 5-caC is converted to a nucleobase which is read as T/ll to a more limited extent.
  • Enzymatic Methyl sequencing involves using T4 bacteriophage p- glucosyltransferase and a TET2 enzyme as the further agents and APOBEC3A as the conversion agent. This process is described in Vaisvila et al. (Genome Res. 2021 , 31 , pp. 1280-1289), US 10,619,200 B2 and US 9,121 ,061 B2, which are incorporated herein by reference.
  • the T4 bacteriophage p-glucosyltransferase converts 5- hydroxymethylcytosine in the target polynucleotide to p-glucosyl-5- hydroxymethylcytosine, which prevents oxidation.
  • the TET2 enzyme causes oxidation of 5-methylcytosine in the target polynucleotide to 5-hydroxymethylcytosine, which in turn is converted to p-glucosyl-5-hydroxymethylcytosine by the T4 bacteriophage p- glucosyltransferase.
  • the TET2 enzyme also causes oxidation of 5-formylcytosine in the target polynucleotide to 5-carboxylcytosine.
  • Subsequent treatment with APOBEC3A converts unmodified cytosines in the target polynucleotide to uracil, as well as 5- carboxylcytosine (including residues that used to be 5-formylcytosine) to a limited extent.
  • EM-seq allows identification of the modified cytosines 5-mC and 5-hmC (as protected glycosyl residues) by reading them as C; whereas unmodified C is converted to U; 5fC and 5-caC are converted to nucleobases which are read as T/U to a limited extent.
  • Modified APOBEC sequencing involves using a mutant APOBEC3A enzyme as the conversion agent, which is described in more detail in the Reference Examples 1 to 4 below. This process is described in US Provisional Application 63/328,444, which is incorporated herein by reference.
  • TAPS TET-assisted pyridine borane sequencing involves using a TET1 enzyme as the further agent and pyridine borane as the conversion agent. This process is described in Liu et al. (Nature Biotechnology, 2019, 37, pp. 424-429), which is incorporated herein by reference.
  • the TET1 enzyme causes oxidation of 5-methylcytosine, 5- hydroxymethylcytosine and 5-formylcytosine in the target polynucleotide to 5- carboxylcytosine.
  • TAPS allows identification of the modified cytosines 5-mC, 5-hmC, 5-fC and 5-caC by reading them as T/ll; whereas unmodified cytosine is read as C.
  • TET-assisted pyridine borane sequencing with p-glucosyltransferase blocking involves using a T4 p-glucosyltransferase and a TET 1 enzyme as the further agents, and pyridine borane as the conversion agent. This process is described in Liu et al. (Nature Communications, 2021 , 12, 618), which is incorporated herein by reference.
  • the T4 p- glucosyltransferase converts 5-hydroxymethylcytosine in the target polynucleotide to p- glucosyl-5-hydroxymethylcytosine, which prevents oxidation.
  • the TET1 enzyme causes oxidation of 5-methylcytosine and 5-formylcytosine in the target polynucleotide to 5- carboxylcytosine.
  • Subsequent treatment with pyridine borane converts 5- carboxylcytosine (including residues that used to be 5-methylcytosine and 5- formylcytosine) to dihydrouracil, but does not convert unmodified cytosine or p-glucosyl- 5-hydroxymethylcytosine.
  • TAPSp allows identification of the modified cytosines 5-mC, 5-fC and 5-caC by reading them as T/U; whereas unmodified cytosine and 5-hmC are read as C.
  • Chemical-assisted pyridine borane sequencing involves using a potassium ruthenate (K2RUO4) as the further agent and 2-picoline borane as the conversion agent. This process is described in Liu et al. (Nature Communications, 2021 , 12, 618), which is incorporated herein by reference. Potassium ruthenate causes oxidation of 5- hydroxymethylcytosine in the target polynucleotide to 5-formylcytosine. Subsequent treatment with 2-picoline borane converts 5-formylcytosine (including residues that used to be 5-hydroxymethylcytosine) and 5-carboxylcytosine to dihydrouracil, but does not convert unmodified cytosine or 5-methylcytosine. Accordingly, CAPS allows identification of the modified cytosines 5-hmC, 5-fC and 5-caC by reading them as T/ll; whereas unmodified cytosine and 5-mC are read as C.
  • K2RUO4 potassium ruthenate
  • Pyridine borane sequencing involves using pyridine borane as the conversion agent. This process is described in Liu et al. (Nature Communications, 2021 , 12, 618), which is incorporated herein by reference. Treatment with pyridine borane converts 5- formylcytosine and 5-carboxylcytosine to dihydrouracil, but does not convert unmodified cytosine, 5-methylcytosine or 5-hydroxymethylcytosine. Accordingly, PS allows identification of the modified cytosines 5-fC and 5-caC by reading them as T/ll; whereas unmodified cytosine, 5-mC and 5-hmC are read as C.
  • Pyridine borane sequencing for 5-caC involves using O-ethylhydroxylamine as the further agent and pyridine borane as the conversion agent. This process is described in Liu et al. (Nature Communications, 2021 , 12, 618), which is incorporated herein by reference.
  • the O-ethylhydroxylamine converts 5-formylcytosine to an oxime derivative, which prevents 5-formylcytosine from converting to dihydrouracil.
  • Subsequent treatment with pyridine borane converts 5-carboxylcytosine to dihydrouracil, but does not convert unmodified cytosine, 5-methylcytosine, 5-hydroxycytosine or the oxime derivative of 5- formylcytosine.
  • PS-c allows identification of the modified cytosine 5-caC by reading it as T/U; whereas unmodified cytosine, 5-mC, 5-hmC and 5-fC are read as C.
  • Also described herein is a method of sequencing at least one polynucleotide sequence to detect modified cytosines, comprising: preparing at least one polynucleotide sequence for detection of modified cytosines using a method as described herein; concurrently sequencing nucleobases in the first portion and the second portion; and identifying modified cytosines by detecting differences when comparing a sequence output from the first portion with a sequence output from the second portion.
  • sequencing is performed by sequencing-by-synthesis or sequencing-by-ligation.
  • the step of preparing the at least one polynucleotide sequence comprises using a selective processing method as described herein; and wherein the step of concurrent sequencing nucleobases in the first portion and the second portion is based on the intensity of the first signal and the intensity of the second signal.
  • the method may further comprise a step of conducting paired-end reads.
  • the method comprises a step of selectively processing the at least one polynucleotide sequence comprising the first portion and the second portion, such that a proportion of first portions are capable of generating a first signal and a proportion of second portions are capable of generating a second signal, wherein the selective processing causes an intensity of the first signal to be greater than an intensity of the second signal
  • the data may be analysed using 16 QAM as mentioned herein.
  • the step of concurrently sequencing nucleobases may comprise:
  • first intensity data comprising a combined intensity of a first signal component obtained based upon a respective first nucleobase at the first portion and a second signal component obtained based upon a respective second nucleobase at the second portion, wherein the first and second signal components are obtained simultaneously;
  • each classification represents a possible combination of respective first and second nucleobases; and (d) based on the selected classification, base calling the respective first and second nucleobases.
  • selecting the classification based on the first and second intensity data may comprise selecting the classification based on the combined intensity of the first and second signal components and the combined intensity of the third and fourth signal components.
  • the plurality of classifications may comprise sixteen classifications, each classification representing one of sixteen unique combinations of first and second nucleobases.
  • the first signal component, second signal component, third signal component and fourth signal component may be generated based on light emissions associated with the respective nucleobase.
  • the light emissions may be detected by a sensor, wherein the sensor is configured to provide a single output based upon the first and second signals.
  • the senor may comprise a single sensing element.
  • the method may further comprise repeating steps (a) to (d) for each of a plurality of base calling cycles.
  • the data may be analysed using 9 QAM as mentioned herein.
  • the step of concurrently sequencing nucleobases may comprise:
  • each classification of the plurality of classifications represents one or more possible combinations of respective first and second nucleobases, and wherein at least one classification of the plurality of classifications represents more than one possible combination of respective first and second nucleobases;
  • selecting the classification based on the first and second intensity data may comprise selecting the classification based on the combined intensity of the first and second signal components and the combined intensity of the third and fourth signal components.
  • an intensity of the first signal component when based on a nucleobase of the same identity, may be substantially the same as an intensity of the second signal component and an intensity of the third signal component is substantially the same as an intensity of the fourth signal component.
  • the plurality of classifications may consist of a predetermined number of classifications.
  • the plurality of classifications may comprise: one or more classifications representing matching first and second nucleobases; and one or more classifications representing mismatching first and second nucleobases, and wherein determining sequence information of the first portion and second portion comprises: in response to selecting a classification representing matching first and second nucleobases, determining a match between the first and second nucleobases; or in response to selecting a classification representing mismatching first and second nucleobases, determining a mismatch between the first and second nucleobases.
  • determining sequence information of the first portion and the second portion may comprise, in response to selecting a classification representing a match between the first and second nucleobases, base calling the first and second nucleobases.
  • determining sequence information of the first portion and the second portion may comprise, based on the selected classification, determining that the second portion is modified relative to the first portion at a location associated with the first and second nucleobases.
  • the first signal component, second signal component, third signal component and fourth signal component may be generated based on light emissions associated with the respective nucleobase.
  • the light emissions may be detected by a sensor, wherein the sensor is configured to provide a single output based upon the first and second signals.
  • the senor may comprise a single sensing element.
  • the method may further comprise repeating steps (a) to (d) for each of a plurality of base calling cycles.
  • Methods as described herein may be performed by a user physically.
  • a user may themselves conduct the methods of preparing at least one polynucleotide sequence for detection of modified cytosines as described herein, and as such the methods as described herein may not need to be computer-implemented.
  • a kit comprising instructions for preparing at least one polynucleotide sequence for detection of modified cytosines as described herein, and/or for sequencing at least one polynucleotide sequence to detect modified cytosines as described herein.
  • the kit may further comprise a sequencing primer comprising or consisting of a sequence selected from SEQ ID NO. 7 to 16 or a variant or fragment thereof.
  • the kit may comprise a sequencing composition comprising a sequencing primer selected from SEQ ID NO. 7 to 10 or a variant or fragment thereof, and a sequencing primer selected from SEQ ID NO. 11 to 16 or a variant or fragment thereof.
  • methods as described herein may be performed by a computer.
  • a computer may contain instructions to conduct the methods of preparing at least one polynucleotide sequence for detection of modified cytosines as described herein, and as such the methods as described herein may be computer-implemented.
  • a data processing device comprising means for carrying out the methods as described herein.
  • the data processing device may be a polynucleotide sequencer.
  • the data processing device may comprise reagents used for synthesis methods as described herein.
  • the data processing device may comprise a solid support, such as a flow cell.
  • a computer program product comprising instructions which, when the program is executed by a processor, cause the processor to carry out the methods as described herein.
  • 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 computer-readable data carrier having stored thereon the computer program product as described herein.
  • DSP digital signal processor
  • ASIC application specific integrated circuit
  • FPGA field programmable gate array
  • a processor can be a microprocessor, but in the alternative, the processor can be a controller, microcontroller, or state machine, combinations of the same, or the like.
  • a processor can also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.
  • systems described herein may be implemented using a discrete memory chip, a portion of memory in a microprocessor, flash, EPROM, or other types of memory.
  • the elements of a method, process, or algorithm described in connection with the embodiments disclosed herein can be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two.
  • 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.
  • Wild type APOBEC3A deaminated 5mC and C substrates to completion, consistent with previous literature. Different mutants exhibited a wide range of reactivities towards 5mC and C substrates, with some showing preference towards either substrate. Remarkably, APOBEC3A(Y130A) (first box) deaminated 5mC substrates almost completely (94.2%), while it deaminated the corresponding C substrate to a minor extent (29.4%). Other mutants, such as APOBEC3A(Y130P) and APOBEC3A(Y130T), also exhibited more complete deamination of the 5mC than C substrate, albeit to a lesser extent than APOBEC3A(Y130A).
  • APOBEC3A(Y130L) (second box) deaminated approximately half of the C substrate (56%), but almost none of the 5mC substrate (6.8%).
  • the deaminase activity of all APOBEC3A(Y130X) mutants is quantified and summarised in the table below:
  • the initial deamination reaction velocity was measured at a range of DNA substrate concentrations and used to construct Michaelis-Menten curves for 5mC and C substrates, respectively.
  • the resulting Km and Kcat values were then derived from these data.
  • the catalytic efficiency of APOBEC3A(Y130A) was ⁇ 100-fold higher on 5mC than C substrates corroborating the endpoint Swal assays shown above.
  • APOBEC3A(Y130A-Y132H) protein was expressed in BL21(DE3) cells, purified using Ni-NTA agarose beads, and desalted/concentrated using spin columns to storage buffer (50mM Tris pH 7.5, 200mM NaCI, 5%(v/v) glycerol, 0.01% (v/v) Tween-20, 0.5mM DTT). This yielded APOBEC3A(Y130A-Y132H) mutant protein preparations with 90-95% purity, as judged by SDS-PAGE analysis.
  • the deaminase activity of purified APOBEC3A(Y130A-Y132H) double mutant protein was then analyzed using the Swal assay, with a 37°C/ 2 hour reaction time and NEB APOBEC3A as positive control.
  • the conditions used were the same as described in Reference Example 2 with the exception that the Swal assay used reaction conditions of 40 mM sodium acetate pH 5.2, 37°C for 1 hour to 16 hours.
  • the DNA substrates are shown below:
  • APOBEC3A (Y130A-Y132H) exhibited higher levels of deamination at all methylated sites compared to unmethylated sites. This was consistent across both CpG and non- CpG contexts, and was robust to variation in reaction time.
  • Biotin-A14-ME (SEQ ID NO. 9)
  • Biotin-TCGT C GGCAGC GT CAGAT GT GTATAAGAGACAG
  • Biotin-B15-ME (SEQ ID NO. 10)
  • transposome complexes were combined together by adding 50pl of each to another 100pl of Standard Storage Buffer to give 200pl of 1 pM transposome mix.
  • Beads were mixed on a rotator for 30mins to 1 hr at room temperature.
  • Beads were resuspended in original volume (200pl) of BLT Storage Buffer (Illumina). The BLTs were stored at 4C until needed.
  • Beads were heated in PAM to 50C for 5mins, then 60C for 5mins.
  • TET master mix (TET MM) was prepared and kept on ice:
  • the mixture was vortexed (or pipette mixed 10x), centrifuged briefly, incubated for 1 hr at 37C, then put on ice.
  • Stop reagent was added, vortexed (or pipette mixed 10x), and incubated at 37C for 30 mins.
  • the 35pl of TET-oxidised DNA from step 8 was combined with 10pl of sodium acetate I acetic acid buffer (pH 4.3) and 5 pl of 1 M pyridine borane. The mixture was incubated overnight at 40C.
  • the mixture was amplified by PCR: cycling procedure - 98C for 30s followed by 3 cycles of (98C 10s, 62C 30s, 65C 3min), then 6 cycles of (98C 10s, 62C 30s, 65C 30s), 65C for 5 mins and then hold at 4C.
  • PCR products were analysed by TapeStation D1000 (Agilent), and then subjected to a further SPRI clean-up before quantification using a Qubit Broad Range dsDNA assay kit (Thermofisher).
  • a custom second hyb was used from the “Cust3” position of the reagent cartridge. This primer hyb maintains a higher temperature (60C) than normal during the post-hyb wash (which usually drops to 40C). This higher temperature was to ensure that the right sequencing primers hyb to the right places on the cluster strands.
  • the primer mix for this custom hyb was HP10 R1 primer mix (Illumina) spiked with 0.5pM each of HYB2’-ME and HYB2-ME primers. These primers are all unblocked and allow concurrent sequencing of both the first portion and the second portion, and so generate the 9 QaM signal during sequencing.
  • the converted library was loaded onto the MiniSeq cartridge at 1 pM final concentration. The MiniSeq was set up to save 3 tiles of images per cycle, for later off-line analysis.
  • the 9 QaM results are shown in Figure 19A, where modified cytosines can be identified by a characteristic central cloud in the plot (indicated by circled region).
  • 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. 25 Uniprot Q9WVE0 MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRY ISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRI FTARLYFCEDRKAEPEGLRR LHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDA FRMLGF
  • SEQ ID NO. 33 Gen Bank XP_045219544.1 MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGFLHNKAKN VPCGDYGCHVELRFLCEVPSWQLDPAQTYRVTWFISWSPCFRKGCAGQVRAFLQENKHVRLRI F AARIYDYDPRYQEALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHSQALSGR LRAILQNQGN
  • SEQ ID NO. 34 GenBank AER45717.1
  • SEQ ID NO. 35 GenBank XP_003264816.1
  • SEQ ID NO. 36 GenBank PNI48846.1
  • SEQ ID NO. 40 Uniprot F6M3K5 MNPQIRNPMERMYRRTFNYNFENEPILYGRSYTWLCYEVKIRKDPSKLPWDTGVFRGQMYSKPE HHAEMCFLSWFCGNQLPAHKRFQITWFVSWTPCPDCVAKVAEFLAEYPNVTLTISAARLYYYWE TDYRRALCRLRQAGARVKIMDYEEFAYCWENFVYNEDQSFMPWYKFDDNYAFLHHKLKEILRHL MDPDTFTSNFNNDLSVLGRHQTYLCYEVERLDNGTWVPMDQHWGFLCNQAKNVPRGDYGCHAEL CFLDQVSSWQLDPAQTYRVTWFISWSPCFSWGCADQVYAFLQENTHVRLRI FAARIYDYNPLYQ EALRTLRDAGAQVSIMTYDEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAILQNQGN

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Abstract

L'invention concerne des procédés de détection de cytosines modifiées dans des séquences d'acides nucléiques.
PCT/EP2023/056668 2022-03-15 2023-03-15 Séquençage simultané de brins complémentaires sens et antisens sur des polynucléotides concaténés pour la détection de méthylation WO2023175040A2 (fr)

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