Classifications

• • 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
• • 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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/154—Methylation markers

Definitions

• This invention relates to the preparation of nucleic acid samples for analysis.
• Single stranded sample preparation is commonly used following bisulfite conversion of DNA molecules.
• the bisulfite conversion process necessarily results in the formation of single stranded DNA, and therefore involves either i) pre-bisulfite sample preparation or ii) post- bisulfite sample preparation employing random priming for downstream analysis.
• Drawbacks to these methods include the potential to generate nicked or fragmented libraries incapable of subsequent amplification, the loss of sequence information from the parent DNA molecules, generation of artefacts that contaminate the sample of interest or induce significant representation bias of reads in the final dataset.
• Bisulfite sequencing allows 5-methylcytosine to be distinguished from the unmethylated cytosine.
• other cytosine modifications including 5- hydroxymethyl and 5-formyl have been identified.
• techniques involving oxidation and/or reduction of the samples prior to bisulfite sequencing have been developed.
• the sequencing output must be compared with a sample which has not undergone bisulfite treatment. Both 5-formylcytosine (5fC) and cytosine (C) are converted to uracil upon bisulfite treatment.
• the inventors have developed a set of molecular tag adaptors allowing the history of a sample to be followed.
• the adaptors have a region having more than one type of cytosine base.
• the use of standard adaptors without different cytosine bases does not allow the history of the sample to be followed as the adaptor sequences are not traceable.
• the tag indexes of the invention on treatment with BS, oxBS or redBS become unique post conversion markers (convertible tags) and show what has happened to the sample.
• the use of multiple tag index sequences allows a plurality of different samples to be analysed in parallel.
• the use a single tag adaptor allows a single library sample to be split into sub aliquots and each sub aliquot processed through a different conversion chemistry.
• the different conversion chemistry causes different conversions to happen to the different cytosine bases within the tag sequence.
• the profile of each molecule in the sequencing run can be determined from the resultant tag sequence, which changes depending on the history of chemical exposure.
• the separately processed samples can thus be pooled together for sequence analysis. Each treated sample can be unambiguously resolved from the pool, and demultiplexed into separate bins determined by sample and conversion type.
• the benefits of the tags include reducing the cost of library construction and sequencing. A single library serves all conversion chemistries, there is no need for any bisulfite specific sample preparation steps. Further advantages include reducing the sources of technical errors and variability induced by the vagaries of library construction. All converted samples share the same, identical starting library, eliminating any library to library construction differences.
• the sequence is a 10-mer with 4 cytosine bases, one of each type (C, mC, hmC and fC).
• C cytosine base
• mC cytosine base
• hmC hmC
• fC cytosine base
• the disclosure includes a nucleic acid adaptor having a tag sequence having at least two different cytosine bases, including one or more modified cytosine bases.
• the tag sequence has a first cytosine base selected from cytosine, 5-methylcytosine, 5-formylcytosine or 5- hydroxymethylcytosine and a second cytosine base has a different nucleotide selected from cytosine, 5-methylcytosine, 5-formylcytosine or 5-hydroxymethylcytosine.
• the term different in this context means chemically or structurally different, not just in an alternative location or of alternative sequence.
• the disclosure includes a nucleic acid adaptor having a tag sequence having at least three chemically different cytosine bases, including two or more modified cytosine bases.
• the tag sequence has a first cytosine base which is unmodified cytosine, a second cytosine base which is 5-methylcytosine, and a third cytosine base which has a nucleotide selected from 5- formylcytosine or 5-hydroxymethylcytosine.
• the term different in this context means chemically or structurally different, not just in an alternative location or of alternative sequence.
• the adaptor may have at least 4 cytosine bases, including 4 chemically different cytosine bases.
• the four different cytosine bases may be a cytosine base, a 5-methylcytosine base, a 5- formylcytosine base and a 5-hydroxymethylcytosine base.
• the tag sequence on the adaptor may be 5-20 bases in length.
• the tag sequence on the adaptor may be 6-12 bases in length.
• the tag sequence may be 10 bases in length.
• the design of the tag may be such that the different cytosine bases are not adjacent in the sequence.
• the tag may be of type C1XC2XC3XC4X where Cl-4 are the different cytosines, and X is one or more nucleotides selected from T, A or G.
• X can be such that the sequence contain a purine (A or G).
• the adaptor can be constructed such that each of the cytosine bases are separated by at least one purine.
• Sequences for the tag sequences may be selected from one or more of the sequences listed below:
• each C is either a cytosine base, a 5-methylcytosine base, a 5- formylcytosine base and a 5-hydroxymethylcytosine base such that each sequence has one of each different type of modification.
• the modifications can be in any order such that any of the C bases can be any modification, providing all four modifications are present in any 10- mer, and no sequence has two identical C bases.
• the tag sequence may be part of a larger oligonucleotide.
• the tagged adaptor may also have a further region for hybridising a primer.
• the larger oligonucleotide adaptor may be part of a single stranded or double stranded adaptor attached to the end of the nucleic acid fragments to be analysed.
• the adaptor sequences may contain methylated C bases instead of C bases. Conventional C nucleotides become uracil bases upon bisulfite treatment, and it may be advantageous to avoid transforming the adaptors with bisulfite.
• the adaptors may therefore contain G, A, T and methyl C bases, with the tag regions only having C, formyl C or hydroxymethyl C bases.
• Examples of types of adaptors carrying tag sequences (shown as 9 or 10-mers in the examples, not to be limited to 9 or 10-mers in reality), are shown in the example below. As can be seen, the adaptors having the tags are larger than just the tags.
• the term tagged adaptors refers to the adaptors having the tag sequences included therein.
• Each cytosine bases is shown as '5' in the sequences above, indicating 5-methylcytosine.
• the adaptors are 'forked adaptors' having a region of double stranded sequence and two regions of single stranded sequence.
• Each adaptor contains a tag in one single stranded region of the 'fork' .
• each adaptor in the example above contains a first strand where every C base is 5-methyl C, and a second strand where every C base except the C bases in the tag is 5-methyl C.
• the term adaptor can apply to either the single stranded oligonucleotides or the hybridised pair of oligonucleotides.
• the invention also includes a nucleic acid sample labelled with a nucleic acid adaptor according as herein described. The sample may be fragmented prior to attachment of the tagged adaptor.
• kits comprising multiple different sequences where each adaptor has two or more different cytosines.
• kits comprising a first nucleic acid adaptor having a tag sequence of 5-20 bases including at least two different cytosine bases in the tag and a second nucleic acid adaptor having a different tag sequence of 5-20 bases including at least two different cytosine bases in the tag.
• the kit may have more than 2 adaptors.
• the kit may have adaptors with at least 4 different tag sequences, each having at least two different cytosine bases in the tag.
• the kit may have adaptors with at least 10 different tag sequences, each having at least two different cytosine bases in the tag.
• the kit may have adaptors with at least 24 different tag sequences, each having at least two different cytosine bases in the tag.
• the sequences of the tags may be selected from the sequences shown above.
• kits comprising multiple different sequences where each adaptor has three or more different cytosines.
• kits comprising a first nucleic acid adaptor having a tag sequence of 5-20 bases including at least three different cytosine bases in the tag and a second nucleic acid adaptor having a different tag sequence of 5-20 bases including at least three different cytosine bases in the tag.
• the kit may have more than 2 adaptors.
• the kit may have adaptors with at least 4 different tag sequences, each having at least three different cytosine bases in the tag.
• the kit may have adaptors with at least 10 different tag sequences, each having at least three different cytosine bases in the tag.
• the kit may have adaptors with at least 24 different tag sequences, each having at least three different cytosine bases in the tag.
• the sequences of the tags may be selected from the sequences shown above.
• the method may include the following steps;
• the method may include additional steps.
• the method may include the step of oxidising and/or reducing the sample prior to bisulfite treatment.
• the bisulfite treatment of the reduced or oxidised sample may take place separately, or with the sample mixed together.
• the method may include the steps of;
• nucleic acid adaptor having at least 4 chemically different cytosine bases, b) fragmenting a nucleic acid sample
• the inventors have developed a set of molecular tag adaptors allowing the chemical exposure history of the molecules in a sample to be followed.
• the adaptors have a region having more than one type of cytosine base.
• the modified region may be the only region on the adaptor having a C base which is not 5-methylcytosine.
• Standard adaptors for use in bisulfite sequencing are usually methylated, and are thus unaffected by bisulfite treatment. Cytosine bases in the adaptor become uracil bases upon bisulfite treatment. Thus the inclusion of both cytosine and methylated cytosine bases in the adaptor attached to a sample, a portion of which undergoes bisulfite treatment, allows identification of whether or not the particular molecules in the sample have undergone bisulfite treatment once the adaptors are sequenced. Similarly the use of hydroxymethylated C or formyl C bases in the adaptor allows identification of whether the samples have been oxidised or reduced prior to bisulfite treatment. The use of all four chemically different cytosine bases in the adaptor attached to a nucleic acid allows the exposure history of the strands in the sample to be followed in a single sequencing run.
• the use of multiple tag index sequences allows a plurality of different samples to be analysed in parallel. Thus for example a number of different samples from different biological origins can be processed in parallel.
• the concept of indexing samples using different molecular sequences on adaptors is known, and can be applied in context. Thus the use of say 24 different adaptors having a different order of A, G, C and T bases, where each of the 24 adaptors has more than one type of C base allows the processing and bisulfite analysis of 24 samples to be achieved in a single sequencing run.
• the concept of indexing is useful in areas where the analysis of small sized genomes is envisaged, for example in sequencing large numbers of microbial samples.
• the use a single tag adaptor allows a single library sample to be split into sub aliquots and each sub aliquot processed through a different conversion chemistry.
• a part of the sample can be oxidised, a further part of the sample reduced, these can be treated with bisulfite along with a further part of the sample, and the bisulfite treated samples can be pooled with a further untreated part and sequenced.
• the different conversion chemistry causes different conversions to happen to the different cytosine bases within the tag sequence. Thus what has happed to each molecule can be seen once the tags are sequenced.
• the separately processed samples can be pooled together for sequence analysis.
• the disclosure includes a nucleic acid adaptor having a tag sequence having at least two different cytosine bases, including one or more modified cytosine bases.
• the disclosure includes an adaptor having A, G, T and 5-methylC bases apart from a tag region where non- methylated C bases (C, HMC or FC bases) are present.
• the tag sequence has a first cytosine base selected from cytosine, 5-methylcytosine, 5-formylcytosine or 5-hydroxymethylcytosine and a second cytosine base has a different nucleotide selected from cytosine, 5- methylcytosine, 5-formylcytosine or 5-hydroxymethylcytosine.
• the term different in this context means chemically or structurally different, not just in an alternative location or of alternate sequence.
• the disclosure includes a nucleic acid adaptor having a tag sequence having at least three chemically different cytosine bases, including two or more modified cytosine bases.
• the tag sequence has a first cytosine base which is unmodified cytosine, a second cytosine base which is 5-methylcytosine, and a third cytosine base which has a nucleotide selected from 5- formylcytosine or 5-hydroxymethylcytosine.
• the term different in this context means chemically or structurally different, not just in an alternative location or of alternative sequence.
• the tag may have at least 4 cytosine bases, including 4 chemically different cytosine bases.
• the four different cytosine bases may be a cytosine base, a 5-methylcytosine base, a 5- formylcytosine base and a 5-hydroxymethylcytosine base.
• the tag sequence on the adaptor may be 5-20 bases in length.
• the tag sequence on the adaptor may be 6-12 bases in length.
• the tag sequence may be 10 bases in length.
• the design of the tag may be such that the different cytosine bases are not adjacent in the sequence.
• the tag may be of type C1XC2XC3XC4X where Cl-4 are the different cytosines, and X is one or more nucleotides selected from T, A or G. X can be such that the gap must contain a purine (A or G).
• the adaptor can be constructed such that the cytosine bases are separated by at least one purine.
• Sequences for the tag sequences may be selected from one or more of the sequences listed below:
• each C is either a cytosine base, a 5-methylcytosine base, a 5- formylcytosine base and a 5-hydroxymethylcytosine base such that each sequence has one of each different type of modification.
• the modifications can be in any order such that any of the C bases can be any modification, providing all four modifications are present in any 10- mer, and no sequence has two chemically identical C bases.
• the tag sequence may be part of a larger oligonucleotide.
• the tagged adaptor may also have a further region for hybridising a primer.
• the larger oligonucleotide adaptor may be part of a single stranded or double stranded adaptor attached to the end of the nucleic acid fragments to be analysed.
• the adaptors may therefore contain G, A, T and methyl C bases, with the tag regions only have C, formyl C or hydroxymethyl C bases.
• the invention also includes a nucleic acid sample labelled with a nucleic acid adaptor according as herein described.
• the sample may be fragmented prior to attachment of the tagged adaptor.
• the population of nucleic acid molecules may be a sample of DNA or RNA, for example a genomic DNA sample.
• Suitable DNA and RNA samples may be obtained or isolated from a sample of cells, for example, mammalian cells such as human cells or tissue samples, such as biopsies.
• the sample may be obtained from a formalin fixed parafin embedded (FFPE) tissue sample.
• FFPE formalin fixed parafin embedded
• the population may be a diverse population of nucleic acid molecules, for example a library, such as a whole genome library or a loci specific library.
• Nucleic acid strands in the population may be amplified nucleic acid molecules, for example, amplified fragments of the same genetic locus or region from different samples.
• Nucleic acid strands in the population may be enriched.
• the population may be an enriched subset of a sample produced by pull-down onto a hybridisation array or digestion with a restriction enzyme.
• the samples having the tagged adaptors may be further processed, for example by amplification or sequencing.
• the joined oligonucleotides may be copied using a nucleic acid polymerase. If adaptors are attached to both ends of the target fragments, the population of fragments can be amplified using a single pair of primers complementary to the adaptors.
• the tags can also be used to help identify sequences from different sources. If adaptors are used with different sequences for different sources of biological materials, then the different sources can be pooled but still identified via the tag when the tags are sequenced. Thus the disclosure herein includes the use of two or more different populations of adaptors for the multiplexing of the analysis of different samples. Disclosed herein therefore are kits containing two or more adaptors of different sequence.
• the sequence of the adaptor oligonucleotide depends on the specific application and suitable adaptor oligonucleotides may be designed using known techniques.
• a suitable adaptor oligonucleotide may, for example, consist of 20 to 100 nucleotides.
• the sequence of the adaptor may be selected to be complementary to a suitable amplification/extension primer.
• the method may be used in order to prepare samples for nucleic acid sequencing.
• the method may be used to sequence a population of synthetic oligonucleotides, for example for the purposes of quality control.
• the first oligonucleotides may come from a population of nucleic acid molecules from a biological sample.
• the population may be fragments of between 100-10000 nucleotides in length.
• the fragments may be 200-1000 nucleotides in length.
• the fragments may be of random variable sequence.
• the order of bases in the sequence may be known, unknown, or partly known.
• the fragments may come from treating a biological sample to obtain fragments of shorter length than exist in the naturally occurring sample.
• the fragments may come from a random cleavage of longer strands.
• the fragments may be derived from shearing the sample using a physical method such as hydrodynamic shearing.
• the fragments may be derived from treating a nucleic acid sample with a chemical reagent (for example sodium bisulfite, acid or alkali) or enzyme (for example with a restriction endonuclease or other nuclease).
• the fragments may come from a treatment step that causes double stranded molecules to become single stranded.
• Methods of the invention may be useful in preparing a population of nucleic acid strands for sequencing, for example a population of bisulfite-treated single- stranded nucleic acid fragments.
• Bisulfite treatment produces single-stranded nucleic acid fragments, typically of about 250-1000 nucleotides in length.
• the population may be treated with bisulfite by incubation with bisulfite ions (HS0 3 2 ).
• bisulfite ions HSU3 2
• the use of bisulfite ions (HSO3 2 ) to convert unmethylated cytosines in nucleic acids into uracil is standard in the art and suitable reagents and conditions are well known.
• the methods disclosed may further include the step of producing one or more copies of the first single stranded oligonucleotides.
• the methods may include producing multiple copies of each of the different sequences.
• the copies may be made by hybridising a primer sequence opposite a universal sequence on the second oligonucleotide sequence, and using a nucleic acid polymerase to synthesise a complementary copy of the first single stranded sequences.
• the production of the complementary copy provides a double stranded polynucleotide.
• the double stranded polynucleotides can be amplified using primers complementary to both strands.
• the amplification can be locus-specific. Locus specific amplification only amplifies a selection of the fragments in the pool and is therefore a selective amplification for certain sequences.
• adaptor sequences can be attached to both ends of the fragments. The attachment of known adaptors at both ends of each fragment can allow amplification of all the fragments in the pool as each fragment possesses two universal ends.
• double stranded polynucleotides may be made circular by attaching the ends together.
• double stranded molecules produced by extension of a primer annealed to the adaptor sequence may be circularised by ligation. This may be useful in the generation of circular nucleic acid constructs and plasmids or in the preparation of samples for sequencing using platforms that employ circular templates (e.g. PacBio SMRT sequencing).
• populations of circularised 3' adapted nucleic acid fragments produced as described herein may be denatured and subjected to rolling circle or whole genome amplification using an amplification primer that hybridises to the 3 '-adaptor oligonucleotide to produce a population of concatomeric products. Amplification of circular fragments can be carried out using primers complementary to two regions of the single adaptor sequence.
• Random priming is used in techniques such as whole genome amplification (WGA). Having a universal primer on one end of a population of single stranded fragments and a random primer on the opposite end means that amplification is more efficient that having random primers on both ends, as is the case with WGA.
• the tagged adaptor joined fragments can be used in any subsequent method of sequence determination.
• the fragments can undergo parallel sequencing on a solid support.
• the attachment of universal adaptors to each end may be beneficial in the amplification of the population of fragments.
• Suitable sequencing methods are well known in the art, and include Illumina sequencing, pyrosequencing (for example 454 sequencing) or Ion Torrent sequencing from Life TechnologiesTM).
• Populations of nucleic acid molecules with a 3' adaptor oligonucleotide and optionally a 5' second adaptor oligonucleotide may be sequenced directly.
• the sequences of the first and second adaptor oligonucleotides may be specific for a sequencing platform.
• they may be complementary to the flowcell or device on which sequencing is to be performed. This may allow the sequencing of the population of nucleic acid fragments without the need for further amplification and/or adaptation.
• the first and second adaptor sequences are different.
• the adaptor sequences and tag sequences are not found within the human genome.
• the nucleic acid strands in the population may have the same first adaptor sequence at their 3' ends and the same second adaptor sequence at their 5' ends i.e. all of the fragments in the population may be flanked by the same pair of adaptor sequences. In such cases both strands in the duplex carries a tag sequence.
• Suitable adaptor oligonucleotides for the production of nucleic acid strands for sequencing may include a region that is complementary to the universal primers on the solid support (e.g. a flowcell or bead) and a region that is complementary to universal sequencing primers (i.e. which when annealed to the adaptor oligonucleotide and extended allows the sequence of the nucleic acid molecule to be read).
• Suitable nucleotide sequences for these interactions are well known in the art and depend on the sequencing platform to be employed. Suitable sequencing platforms include Illumina TruSeq, LifeTech IonTorrent, Roche 454 and PacBio RS.
• the sequences of the first and second adaptor oligonucleotides may comprise a sequence that hybridises to complementary primers immobilised on the solid support (e.g. 20- 30 nucleotides); a sequence that hybridises to sequencing primer (e.g. 30-40 nucleotides) and a unique index sequence (e.g. 6-10 nucleotides).
• Suitable first and second adaptor oligonucleotides may be 56-80 nucleotides in length.
• the adaptors may be configured as single strands containing both DNA and RNA, or as two or three strands.
• the nucleic acid molecules may be purified by any convenient technique. Following preparation, the population of nucleic acid molecules may be provided in a suitable form for further treatment as described herein. For example, the population of nucleic acid molecules may be in aqueous solution in the absence of buffers before treatment as described herein.
• populations of nucleic acid molecules with a 3' adaptor oligonucleotide and optionally a 5' adaptor oligonucleotide may be further adapted and/or amplified as required, for example for a specific application or sequencing platform.
• the nucleic acid strands in the population may have the same first adaptor sequence at their 3' ends and the same second adaptor sequence at their 5' ends i.e. all of the fragments in the population may be flanked by the same pair of adaptors, as described above. This allows the same pair of amplification primers to amplify all of the strands in the population and avoids the need for multiplex amplification reactions using complex sets of primer pairs, which are susceptible to mis-priming and the amplification of artefacts.
• Suitable first and second amplification primers may be 20-25 nucleotides in length and may be designed and synthesised using standard techniques.
• a first amplification primer may hybridise to the first adaptor sequence i.e. the first amplification primer may comprise a nucleotide sequence complementary to the first adaptor oligonucleotide; and a second amplification primer may hybridises to the complement of second adaptor sequence i.e. the second amplification primer may comprise the nucleotide sequence of the second adaptor oligonucleotide.
• a first amplification primer may hybridise to the complement of first adaptor sequence i.e.
• the first amplification primer may comprise a nucleotide sequence of the first adaptor oligonucleotide; and a second amplification primer may hybridise to the second adaptor sequence i.e. the second amplification primer may comprise the nucleotide sequence of the second adaptor oligonucleotide.
• the first and second amplification primers may incorporate additional sequences. Additional sequences may include index sequences to allow identification of the amplification products during multiplex sequencing, or further adaptor sequences to allow sequencing of the strands using a specfic sequencing platform.
• a portion of the nucleic acid sample may be oxidised using an oxidising agent.
• the oxidising agent may be a non-enzymatic oxidising agent, for example, an organic or inorganic chemical compound.
• Suitable oxidising agents are well known in the art and include metal oxides, such as KRu0 4 , Mn0 2 and KMn0 4 .
• Particularly useful oxidising agents are those that may be used in aqueous conditions, which are most convenient for the handling of the polynucleotide. However, oxidising agents that are suitable for use in organic solvents may also be employed where practicable.
• the oxidising agent may comprise a perruthenate anion (Ru0 4 ).
• Suitable perruthenate oxidising agents include organic and inorganic perruthenate salts, such as potassium perruthenate (KRu0 4 ) and other metal perruthenates; tetraalkyl ammonium perruthenates, such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP); polymer-supported perruthenate (PSP) and tetraphenylphosphonium ruthenate.
• the oxidising agents may be a metal (VI) oxo complex.
• the oxidising agent may be manganate (Mn(VI)0 4 2" ), ferrate (Fe(VI)0 4 2" ), osmate (Os(VI)0 4 2” ), ruthenate (Ru(VI)0 4 2” ), or molybdate (Mo(VI)0 4 2" ).
• the oxidising agent or the oxidising conditions may also preserve the polynucleotide in a denatured state.
• the polynucleotides in the first portion may be purified.
• nucleic acid purification may be performed using any convenient nucleic acid purification technique. Suitable nucleic acid purification techniques include spin-column chromatography.
• the polynucleotide may be subjected to further, repeat oxidising steps. Such steps are undertaken to maximise the conversion of 5-hydroxycytosine to 5-formylcytosine. This may be necessary where a polynucleotide has sufficient secondary structure that is capable of re- annealing. Any annealed portions of the polynucleotide may limit or prevent access of the oxidising agent to that portion of the structure, which has the effect of protecting 5-hydroxycytosine from oxidation.
• the portion of the population of polynucleotides may for example be subjected to multiple cycles of treatment with the oxidising agent followed by purification. For example, one, two, three or more than three cycles may be performed.
• a portion of the population of polynucleotides comprising the sample nucleotide sequence may be reduced. In other embodiments, a further portion of the population of polynucleotides comprising the sample nucleotide sequence may be reduced.
• Reduction converts 5-formylcytosine residues in the sample nucleotide sequence into 5- hydroxymethylcytosine.
• the portions of polynucleotides may be reduced by treatment with a reducing agent.
• the reducing agent is any agent suitable for generating an alcohol from an aldehyde.
• the reducing agent or the conditions employed in the reduction step may be selected so that any 5-formylcytosine is selectively reduced (i.e.
• the reducing agent or reduction conditions are selective for 5-formylcytosine). Thus, substantially no other functionality in the polynucleotide is reduced in the reduction step.
• the reducing agent or conditions are selected to minimise or prevent any degradation of the polynucleotide.
• Suitable reducing agents are well-known in the art and include NaBH 4 , NaCNBH 4 and LiBH . Particularly useful reducing agents are those that may be used in aqueous conditions, as such are most convenient for the handling of the polynucleotide. However, reducing agents that are suitable for use in organic solvents may also be employed where practicable.
• the reduced and oxidised portion of the population are treated with bisulfite.
• a second portion of the population which has not been oxidised or reduced is also treated with bisulfite.
• the bisulfite treatment can be done separately on the three samples, or the samples can be pooled so that the reduced, oxidised and untreated sample and all exposed to bisulfite in the same reaction.
• Bisulfite treatment converts both cytosine and 5-formylcytosine residues in a polynucleotide into uracil. Where any 5-carboxycytosine is present (as a product of the oxidation step), this 5-carboxycytosine is converted into uracil in the bisulfite treatment. Without wishing to be bound by theory, it is believed that the reaction of the 5-formylcytosine proceeds via loss of the formyl group to yield cytosine, followed by a subsequent deamination to give uracil. The 5-carboxycytosine is believed to yield the uracil through a sequence of decarboxylation and deamination steps. Bisulfite treatment may be performed under conditions that convert both cytosine and 5-formylcytosine or 5-carboxycytosine residues in a polynucleotide as described herein into uracil.
• a portion of the population may be treated with bisulfite by incubation with bisulfite ions (HSO3 2 ).
• bisulfite ions HSO3 2
• the use of bisulfite ions (HSO3 2 ) to convert unmethylated cytosines in nucleic acids into uracil is standard in the art and suitable reagents and conditions are well known to the skilled person. Numerous suitable protocols and reagents are also commercially available (for example, EpiTect , Qiagen L; EZ DNA Methyl ationTM Zymo Research Corp CA; CpGenome Turbo Bisulfite Modification Kit; Millipore).
• kits comprising multiple different sequences where each adaptor has two or more different cytosines.
• kits comprising a first nucleic acid adaptor having a tag sequence of 5-20 bases including at least two different cytosine bases in the tag and a second nucleic acid adaptor having a different tag sequence of 5-20 bases including at least two different cytosine bases in the tag.
• the kit may have more than 2 adaptors.
• the kit may have adaptors with at least 4 different tag sequences, each having at least two different cytosine bases in the tag.
• the kit may have adaptors with at least 10 different tag sequences, each having at least two different cytosine bases in the tag.
• the kit may have adaptors with at least 24 different tag sequences, each having at least two different cytosine bases in the tag.
• the sequences of the tags may be selected from the sequences shown below:
• each C base in each tag is different; one is C, one is methyl C, one is hydroxymethyl C and one is formyl C.
• the kits may contain one of more nucleic acid acting enzymes such as nucleic acid polymerases or ligases.
• the adapter kits may contain a further nucleic acid complementary to a region of the first adapter such that the adaptor is at least partly double stranded.
• the tag sequences may be selected using the protocol below, or modifications thereto:
• Each tag must contain at least 4 cytosines to represent each modification (C, mC, hmC, fC).
• the tags may be incorporated into adaptors used to attached to nucleic acid fragments for sequencing.
• a method of using the tagged adaptors for determining the methylation profile of a nucleic acid sample may include the following steps; a) preparing a nucleic acid adaptor having a tag sequence having at least two cytosine bases, including one or more modified cytosine bases, wherein a first cytosine base has a nucleotide selected from cytosine, 5-methylcytosine, 5-formylcytosine or 5- hydroxymethylcytosine and a second cytosine base has a different nucleotide selected from cytosine, 5-methylcytosine, 5-formylcytosine or 5-hydroxymethylcytosine,
• the method may include additional steps.
• the method may include the step of oxidising and/or reducing the sample prior to bisulfite treatment.
• the bisulfite treatment of the reduced or oxidised sample may take place separately, or with the sample mixed together.
• the method may include the steps of;
• nucleic acid adaptor having at least 4 chemically different cytosine bases, b) fragmenting a nucleic acid sample
• the method may include the step of oxidising the sample prior to bisulfite treatment.
• the method may include the steps of;
• the oxidising agent may be a non-enzymatic oxidising agent, for example, an organic or inorganic chemical compound.
• Suitable oxidising agents are well known in the art and include metal oxides, such as KRu0 4 , Mn0 2 and KMn0 4 .
• Particularly useful oxidising agents are those that may be used in aqueous conditions, which are most convenient for the handling of the polynucleotide.
• oxidising agents that are suitable for use in organic solvents may also be employed where practicable. In such cases the three different cytosine bases may be cytosine, 5-methylcytosine and 5-hydroxymethylcytosine.
• the oxidising agent may comprise a perruthenate anion (Ru0 4 ).
• Suitable perruthenate oxidising agents include organic and inorganic perruthenate salts, such as potassium perruthenate (KRu0 4 ) and other metal perruthenates; tetraalkyl ammonium perruthenates, such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP); polymer-supported perruthenate (PSP) and tetraphenylphosphonium ruthenate.
• the oxidising agents may be a metal (VI) oxo complex.
• the oxidising agent may be manganate (Mn(VI)0 4 2" ), ferrate (Fe(VI)0 4 2” ), osmate (Os(VI)0 4 2” ), ruthenate (Ru(VI)0 4 2” ), or molybdate (Mo(VI)0 4 2" ).
• the method may include the step of reducing the sample prior to bisulfite treatment. The method may include the steps of;
• the reducing agent may be borohydride.
• Suitable reducing agents include NaBH 4 , NaCNBH 4 and LiBH 4 .
• the three different cytosine bases may be cytosine, 5- methylcytosine and 5-formylcytosine.
• CEG04 41 4 400 ng each) and one third (CEG04 41 4) was left native, one third (CEG04 41 5) was converted through the bisulfite only half of the CEGX TrueMethyl kit (as per manufacturers protocol) and a third (CEG04 41 6) was converted through the oxidative bisulfite half of the CEGX TrueMethyl kit. (as per manufacturers protocol). All samples were amplified using the PCR protocol in the TrueMethyl kit using the TrueMethyl polymerase. Amplicons were quantified and pooled at equimolar concentrations and used to prepare a 2nM library solution to take forward for Illumina SBS sequencing.
• This experiment demonstrates the use of convertible index tags to uniquely differentiate between samples derived from a common library processed using different conversion chemistries. Reads processed through BS, oxBS or untreated can be unambiguously deconvolved from a complex pool of indexed fragments.
• Table 2 Summary of the mapping efficiencies of the Native, BS and oxBS treated samples

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