WO2016170319A1 - Enrichissement d'échantillons d'acide nucléique - Google Patents

Enrichissement d'échantillons d'acide nucléique Download PDF

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WO2016170319A1
WO2016170319A1 PCT/GB2016/051083 GB2016051083W WO2016170319A1 WO 2016170319 A1 WO2016170319 A1 WO 2016170319A1 GB 2016051083 W GB2016051083 W GB 2016051083W WO 2016170319 A1 WO2016170319 A1 WO 2016170319A1
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adaptor
locus specific
primers
specific primers
sample
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PCT/GB2016/051083
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Neil Matthew BELL
Andreas Claas
Tobias William Barr Ost
Shirong YU
Floriana MANODORO
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Cambridge Epigenetix Ltd
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • This invention relates to the preparation of nucleic acid samples for analysis and certain methods and tools for the selection of specific regions of interest from a nucleic acid sample.
  • Single stranded sample preparation is commonly required 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.
  • a direct method of ligating the termini of single stranded DNA post-bisulfite treatment in quantitative yield is of significant interest.
  • Targeted enrichment is a method used commonly in genomic and epigenomic analysis to reduce the complexity of the genome being studied and to home-in on specific regions of interest (e.g. exomes, CpGs, specific genes etc). This allows the cost of sequencing to be decreased dramatically and the complexity of analysis simplified.
  • Targeted enrichment has been shown to be an effective and reliable alternative method to whole genome analysis in situations where only a fraction of the genome needs to be interrogated or where experiments are done at such scale (numbers of samples) that to do sequencing analysis at a whole genome scale becomes cost prohibitive.
  • pairs of primers for each amplicon, or locus are required and this can limit the complexity (number of locus amplicons targeted) of a single enrichment event; also if using primers with a 5'-flap non-complementary to the loci but complementary to the sequencing platform intended for analysis, this can simplify the workflow but decrease primer specificity, increase cost of the targeting primers and decrease the complexity of targeting.
  • hybridisation arrays or in-solution capture methods typically these target adapted fragments prepared from double stranded (ds) DNA and as such limit their usefulness to native DNA applications. Methods do exist that allow targeted enrichment of bisulfite converted DNA (e.g.
  • hybridisation methods need two pairs of primers to get full information from the same region.
  • Methods described herein can utilise a single primer per loci (not a pair) which should allow improvements in specificity and enable more loci to be targeted within a single reaction. Only 2 primers per region will give information on the top and bottom converted strand of a selected region.
  • hybridisation pull-down methods are inefficient. Working with low mass samples can be a problem simply due to liquid handling losses and multiple transfer steps.
  • a small subset of fragments e g the 1-2 % of the genome represented by the exome
  • the vast majority of the sample remains in solution, so the amount of material required as the input sample is high as most of the sample is not captured.
  • Elution of captured DNA from beads is also inefficient. Methods described herein can be used as a one-pot reaction that should offset these issues and will not be reliant on bead purifications and pulldowns.
  • the IlluminaTM Human Methylation 450K Array is an example of a targeted enrichment array format that is well utilised by the epigenetics research community, but has several drawbacks. It is limited to a physical array format which yields non-digital data and makes sample multiplexing at high number cumbersome and dependent on sophisticated automation. The method is only compatible with Human samples, limiting its usefulness. The number of probes and their specificity is high (-480,000) but the method relies on whole genome amplification using random priming and as such is potentially biased.
  • Methods described herein can be sequencing based and so would yield digital data, are agnostic to species (target primer pools could be designed to any species) and there is less inertia required to prepare multiple different targeting pools than is the case for array-based techniques.
  • Sample multiplexing can be significantly simpler, and uses approaches common in the art such as molecular barcodes (tags) attached to identify different populations of primers.
  • PCR is inherently biased, particularly when amplifying bisulfite converted (AT-rich) DNA. Data generated by methods dependant on PCR will necessarily be of lower quality and more biased than those which no not employ PCR. Methods described herein method would work with or without PCR, so not only can the effect of PCR be evaluated, it can be eliminated as a source of doubt in the targeting experiments.
  • Amplification of regions of greater than around 400bp from bisulfite converted DNA is difficult due to the fragmentation induced by the bisulfite conversion process. This fragmentation is random, but decreases the molarity of fragments in the pool that remain intact in the region of interest. Higher concentration of template is typically required in the amplicon reactions in order to account for this, which decreases the specificity of targeting and increases the sample mass burden required per amplicon reaction (which can be a problem for precious samples). The same is true for methods that depend on targeting a converted adapted NGS library. The conversion process fragments the library, decreasing the molarity of intact fragments (DNA inserts flanked by two universal primer regions) which decreases the diversity (number of unique fragments) in a given sample.
  • Methods described herein allow for the targeted enrichment of converted, single stranded fragments derived directly from genomic DNA or alternative samples without the necessity for PCR or adaption of the samples.
  • the method relies on the direct hybridisation of locus specific primers with the fragmented sample and extension of the primers hybridised to the sample. Only the fragments arising from the successful hybridisation and extension of the loci-specific primers are then analysed further, improving the specificity of targeting and simplifying the complexity of the targeting selection and isolation. Further benefits include an increase in the number of loci specific probe oligos that can be used in a single targeting pool compared to alternative methods, allowing more features to be targeted, generating a richer dataset with improved resolution and quality.
  • Methods described herein will yield a significant competitive advantage in data quality and cost per data point in the current epigenomic marketplace.
  • the method can work robustly with millions of loci targeting probes.
  • Disclosed is a method of selecting and analysing a subset of a nucleic acid sample comprising;
  • sample fragments do not have an adaptor attached prior to hybridising the population of locus specific primers.
  • the sample fragments contain only biological source material, there are no artificial sequences added prior to the hybridisation and extension steps.
  • Fragmentation of the sample can be caused by bisulfite treatment.
  • Bisulfite treatment converts non-methylated cytosine bases to uracil bases.
  • the locus specific region can contain only the nucleic acid bases A, C and T such that the locus is complementary to a sample treated with bisulfite.
  • the locus can contain all four bases.
  • the population of locus specific primers can contain both primers with A, C and T and primers with A, C, G and T. Primers having both G and T in equivalent positions can be used to identify specific methylation sites if required. Alternatively the methylation sites of interest may be in the extension region adjacent to the 3 ' end of the primer.
  • the method can work robustly with millions of loci targeting probes.
  • the population of locus specific primers can be at least 50 different sequences.
  • the population of locus specific primers can be at least 100 different sequences.
  • the population of locus specific primers can be at least 1000 different sequences.
  • the population of locus specific primers can be at least 10,000 different sequences.
  • the population of locus specific primers can be at least 100,000 different sequences.
  • the population of locus specific primers can be at least 1,000,000 different sequences.
  • the population of primers can contain a universal region common to all primers.
  • the universal region is preferably not complementary to the sample of interest.
  • the universal region can be located at the 5' end of the primer.
  • the non-sample complementary region may include an identifier region or tag to enable sample multiplexing.
  • Disclosed is a method of selecting and analysing a subset of a nucleic acid sample comprising;
  • the extension reaction(s) may be carried out using a nucleic acid polymerase.
  • the extension reaction(s) may be carried out using nucleotide triphosphates.
  • the extension can be carried out using four nucleotide triphosphates. If desired, the reaction may be carried out without the presence of dGTP, thereby removing fragments containing residual cytosine bases. In the case of bisulfite treated samples, the methylated cytosine fragments can be effectively removed by carrying out the extension in the presence of dATP, dCTP and dTTP (or dUTP). In such cases the extension is terminated when a methyl C is reached.
  • the non- methyl C can incorporate a dATP, hence allowing continued extension.
  • the first extension can be carried out using dUTP.
  • the dUTP can either replace dTTP or be used alongside dTTP, thus the nucleotides can be dUTP, dATP, dCTP and optionally dGTP and optionally dTTP.
  • the use of dUTP instead of or alongside dTTP allows the extended strand to be subsequently completely or partially removed by digestion if desired.
  • Using a mixture of dUTP and dTTP allows the strands to be randomly cleaved in different locations, and hence different length extension products can be analysed (akin to extension ladders in conventional Sanger sequencing).
  • the hybridisation and extension cycles can be repeated.
  • the primer hybridisation can be carried out in a single cycle, or the locus specific hybridisation and subsequent extension can be repeated, for example using thermocycling.
  • the extended strand should terminate in a 3 '-hydroxyl group.
  • the extended primers have a newly generated 3 ' end, the 3 ' hydroxyl group resulting from an incorporated nucleotide (from the dNTP).
  • the 3' hydroxyl group in the extended strand can be attached to an adaptor.
  • the adaptor can be attached using a ligase.
  • the adaptor can be attached using a polymerase.
  • the polymerase may be a template independent polymerase.
  • the template independent polymerase can be terminal transferase (TdT).
  • the adaptor can contain a triphosphate moiety.
  • the adaptor can contain a region of known sequence, allowing amplification of the adapted extension primers.
  • the adapted extension primers can be copied by hybridising a second primer to the adaptor and extending the second primer to produce copies of the fragments in a second extension.
  • the adaptor may be a hairpin, whereby the extension can be carried out from the 3 ' end of the hairpin.
  • the resulting extended products are the only species in the mixture having a blunt, double stranded end.
  • the double stranded end can undergo further modification in order to attach an adaptor.
  • the excess primers can be removed, for example by digestion of the single stranded products. If the unextended primers are not removed, then adaptors may attach directly to the primers, resulting in adaptor-dimer products with no sample fragments between the adaptors.
  • the locus primers are single stranded, whereas the extended primers are double stranded.
  • methods for the removal of single stranded primers from double stranded fragments can be used, for example nuclease digestion.
  • the primers may contain one of more ribonucleotides in order to facilitate digestion.
  • the primers may be RNA primers, thus enabling complete removal of the single stranded RNA primers, whilst leaving the RNA/DNA hybrid duplexes intact. If the extended products are single stranded, as for example those obtained from multiple cycles of extension, the products may still be digested if the ends of the primers are susceptible to digestion, but the ends of the extension products are not. For example if the ends of the primers contain a ribonucleotide, but the extension products are deoxyribonucleotides then the primers can be digested.
  • the primers can contain a modification which can undergo extension with nucleotide triphosphates, but can not undergo attachment to an adaptor, thereby enabling the extension material to be solely adapted.
  • nucleotide triphosphates are used during the extension reaction.
  • the nucleotide extension products contain a thiophosphate backbone which would be protected from digestion.
  • one or more of the nucleotide triphosphates could contain a ligand such as biotin, thereby allowing for physical separation of the extended products from the unextended primers which do not contain the ligand.
  • a primer may be hybridised to the adaptor to enable a second extension.
  • the adaptor may be a self-priming hairpin.
  • the second extension reaction may be carried out using a nucleic acid polymerase.
  • the extension reaction may be carried out using nucleotide triphosphates.
  • the extension can be carried out using four nucleotide triphosphates.
  • the four nucleoside triphosphates may be dCTP, dATP, dGTP and dTTP.
  • the use of dTTP allows strands made using dUTP in the first extension to be cleaved, leaving solely the second extension strands intact.
  • the extension strands may be treated to remove the uracil bases, thereby allowing selective strand cleavage.
  • the extension strands may be treated with the enzyme UDG, resulting in abasic sites, which can then be cleaved using an endonuclease,
  • the second extension results in products having a 3 '-hydroxyl group. If the locus primers have a known universal region, then the ends of each of the products from the second extension has a sequence complementary to the universal region.
  • the 3 ' hydroxyl group in the extended strand can be attached to an adaptor.
  • the adaptor can be attached using a ligase.
  • the adaptor can be attached using a polymerase, for example a template independent polymerase.
  • the template independent polymerase can be terminal transferase (TdT).
  • TdT terminal transferase
  • the method results in products having known regions at either end and copies of the nucleic acid sample fragments centrally between the known ends.
  • the copied fragments can therefore be amplified using primers complementary to the two adaptors/universal regions.
  • the sequencing step can be carried out on the amplified mixture.
  • the adaptor may contain a triphosphate moiety.
  • the triphosphate moiety may attach to the 3 '- hydroxyl of the extended primer strand.
  • the triphosphate moiety may be attached to the 5'- end of the nucleic acid adaptor via a linker.
  • the linker may contain a nucleotide having a ribose or deoxyribose moiety, with the oligonucleotide adaptor attached via the nucleotide base.
  • the adaptor, or oligonucleotide 5 '-triphosphate adaptor may be single stranded or double stranded.
  • the double stranded adaptor has at least one overhanging single stranded region, and may have two or three overhanging single stranded regions. At least one of the overhangs serves to act to hybridise to the end of the extended locus primers to which the adaptor is to be attached, and acts as a site which can undergo polymerase extension to make the attached single stranded extended locus primers double stranded.
  • the adaptors can be 'forked' adaptors having regions which are non-complementary as well as regions which are complementary.
  • the adaptor may take the form of a hairpin.
  • a hairpin is a nucleic acid sequence containing both a region of single stranded sequence (a loop region) and regions of self-complementary sequence such that an intra-molecular duplex can be formed under hybridising conditions (a stem region).
  • the stem may also have a single stranded overhang.
  • the primer can be part of a mixture of primers, each primer having a locus specific region at the 3 ' end and a universal region at the 5' end.
  • Disclosed herein is a method of joining a first single stranded oligonucleotide and an oligonucleotide adaptor using a template independent nucleic acid polymerase enzyme, wherein the first single stranded oligonucleotide is obtained by extending a primer and the second oligonucleotide adaptor takes the form of a hairpin having a single stranded region and a region of self-complementary double stranded sequence capable of forming a duplex under hybridising conditions.
  • the hairpin adaptor has an extendable 3' end.
  • extension of the 3 ' end allows copying of the adapted fragments.
  • the method may include a step of using a nucleic acid polymerase to extend the 3 '-end of the oligonucleotide adaptor to produce a copy of the extended locus specific primers.
  • the first single stranded oligonucleotides are fragments derived from a nucleic acid sample.
  • the fragments can be obtained using chemical or enzymatic cleavage of the sample.
  • the fragments can be obtained using bisulfite treatment.
  • the locus and extension may give rise to fragments of say 100-200 bases. Attachment of the adaptor, followed by extension gives rise to products having say 100-200 base pairs of double stranded sequence, linked at one end by a loop of single stranded sequence from the hairpin adaptor.
  • the extension should give rise to a blunt-ended product, including the complement of the locus primer and any universal region attached thereto.
  • a further adaptor can be attached to the end of the extended copy.
  • the universal region and/or further adaptor results in products having known regions at either end, and copies of the nucleic acid sample fragments centrally between the known ends.
  • the copied fragments can therefore be amplified using primers complementary to the two adaptors or copies thereof.
  • the sequencing step can be carried out on the amplified mixture.
  • each member of the population contains a common universal sequence and one of a plurality of locus-specific regions wherein each locus specific region contains only the nucleic acid bases A, C and T such that the locus is complementary to a sample treated with bisulfite.
  • Bisulfite treatment results in a sample having very few residual C bases, and so the primers can be free of the G nucleotide.
  • the primer can contain G bases in the universal region, but the primer regions which are locus specific and which vary between different members of the population can be G-free (i.e. contain only A, C and T).
  • kits for use in selecting fragments from a nucleic acid sample comprising a plurality of locus specific primers, a hairpin polynucleotide having a triphosphate moiety at the 5 '-end and a terminal transferase.
  • Other components, including instructions, can be added to the kit as described herein.
  • the kit may contain the plurality of locus specific primers as described above.
  • the method herein describes a number of features different to prior art methods of nucleic acid selection. These include:
  • the nucleic sample is fragmented as a first part of the process.
  • the sample may be the native biological sample (for example raw genomic DNA).
  • the sample has not undergone any amplification or adaptor attachment steps, so the potential for selection or amplification bias is reduced.
  • the locus specific primers are hybridised directly to the fragmented sample. There is therefore no possibility of mis-priming to portions of any adaptors, as no adaptors are present at the point the primers are hybridised.
  • Disclosed is a method of selecting a subset of a nucleic acid sample comprising;
  • steps a-e should be carried out in the order shown.
  • the primers are loci specific, meaning that they hybridise to a single location in the nucleic acid sample of interest.
  • the primers can be of different lengths in order to normalise melting temperatures.
  • the primers can be between 15-40 bases in length.
  • Fragmentation of the sample can be caused by bisulfite treatment.
  • Bisulfite treatment converts non-methylated cytosine bases to uracil bases.
  • the locus specific region can contain only the nucleic acid bases A, C and T such that the locus is complementary to a sample treated with bisulfite.
  • the locus can contain all four bases.
  • the population of locus specific primers can contain both primers with A, C and T and primers with A, C, G and T. Primers having both G and T in equivalent positions can be used to identify specific methylation sites if required. Alternatively the methylation sites of interest may be in the extension region adjacent to the 3 ' end of the primer.
  • the locus specific primers can be chosen bioinformatically to cover regions of interest.
  • the primers can be chosen to locate near to CpG islands or potential methylation sites of interest.
  • Extension of the primers can be chosen to read through one or more CpG locations.
  • each primer can consist of A, C and T bases in the locus specific region, and A, C, G and T bases in the universal region.
  • the universal region should be to the 5' side of the locus region such that 3 ' end of the primer is hybridised and suitable for extension.
  • Each locus can be close to a CpG location in the sample.
  • the length of the extension products is generally determined by the length of the fragments and the position of hybridisation. The extension will continue either until the end of the template is reached or a site is reached which does not permit incorporation, either for example because the nucleoside is abasic, or a limited selection of nucleoside triphosphates (less than 4) is used. The length of the extension is not particularly significant, and can be for example on average 10-200 bases per molecule.
  • the extension reaction may be carried out using a nucleic acid polymerase.
  • the extension reaction may be carried out using nucleotide triphosphates.
  • the extension can be carried out using four nucleotide triphosphates.
  • the reaction may be carried out without the presence of dGTP, thereby removing fragments containing residual cytosine bases.
  • the methylated cytosine fragments can be effectively removed by carrying out the extension in the presence of dATP, dCTP and dTTP (or dUTP).
  • the extension is terminated when a methyl C is reached.
  • the non- methyl C (converted to U) can incorporate a dATP, hence allowing continued extension.
  • the first extension can be carried out using dUTP.
  • the dUTP can either replace dTTP or be used alongside dTTP, thus the nucleotides can be dUTP, dATP, dCTP and optionally dGTP and optionally dTTP.
  • the use of dUTP instead of or alongside dTTP allows the extended strand to be subsequently completely or partially removed by digestion if desired.
  • Using a mixture of dUTP and dTTP allows the strands to be randomly cleaved in different locations, and hence different length extension products can be analysed (akin to extension ladders in conventional Sanger sequencing).
  • the extension can be optimised in order to optimise the amount of information obtained from the sequencing process.
  • the extension may be carried out with less than four dNTP's.
  • the extension may be carried out with two or three dNTP' s.
  • the reactions may be carried out with one or more rNTP' s.
  • the extension may be carried out with one rNTP and three dNTP' s. In such cases the extension may be inhibited as a DNA polymerase is unable to incorporate a ribo-NTP, and thus extension is prevented in a similar way to the absence of the corresponding dNTP.
  • the extension process can be carried out via thermocycling such that multiple extension products are derived from each fragment.
  • Alternative isothermal processes of denaturation and re-annealing can also be carried out such that the extended products are removed, and fresh primers are hybridised.
  • the nucleotide triphosphates can be modified (non-natural). Modifications may allow for the extended strands to be isolated from non-extended primers, or may allow for protection from digestion. Modifications may include for example a biotin moiety or a phosphorothioate moiety.
  • the location of the base(s) being analysed is determined by the identity of a specific primer. More than one base can be analysed per extended primer.
  • the extension products an be used to analyse nucleotide changes, for example single nucleotide polymorphisms (SNP's) or methylation status, for example whether C cases have been converted to U upon bisulfite treatment.
  • SNP's single nucleotide polymorphisms
  • methylation status for example whether C cases have been converted to U upon bisulfite treatment.
  • the multiple base extensions can give information in relation to deletions or insertions of one or more bases.
  • loci specific or locus specific means that the primer hybridises selectively to a single location, or loci, in the nucleic acid sample.
  • the method can be carried out using a large number of different primers which can be pooled prior to hybridisation with the sample. There is no particular limit to the number of loci analysed. The method can work robustly with millions of loci targeting probes.
  • the population of locus specific primers can be at least 50 different sequences.
  • the population of locus specific primers can be at least 100 different sequences.
  • the population of locus specific primers can be at least 1000 different sequences.
  • the population of locus specific primers can be at least 10,000 different sequences.
  • the method can be carried out such that at least 100,000 locations (primers) are analysed per sample.
  • the method can be carried out such that at least 200,000 locations (primers) are analysed per sample.
  • the method can be carried out such that at least 300,000 locations (primers) are analysed per sample.
  • the method can be carried out such that at least 400,000 locations (primers) are analysed per sample.
  • the population of locus specific primers can be at least 1,000,000 different sequences.
  • the location(s) in the sample to be identified is/are in the vicinity of a unique primer.
  • the base(s) to be interrogated should be at the 3 '- side of the primer such that nucleotides can be incorporated complementary to the base(s) being analysed.
  • the base(s) to be interrogated may be immediately 3 ' of the primer such that the first incorporation is being studied, or may be within 2-30 bases of the end of the primer.
  • the interrogated bases can be in different locations for different primers.
  • the primers can be of different lengths in order to normalise melting temperatures.
  • the primers can be between 15-40 bases in length. Primers having higher levels of A and T bases can be longer than primers having higher levels of C and G bases.
  • the primers should be specifically hybridised at the temperature required for the polymerase extension.
  • the primers can be extended using a suitable nucleic acid polymerase.
  • the polymerase may be a DNA polymerase.
  • the polymerase may be active at room temperature, or may be a thermophilic polymerase.
  • the temperature of the extension reaction can be chosen based on the desired specificity of the primer hybridisation reactions and the length of the primer sequences.
  • the temperature of the extension reaction can be for example between 30-72 °C.
  • the temperature of the extension reaction can be for example between 50-72 °C.
  • the nucleic acid samples are prepared as single stranded, which are then hybridised with the primers.
  • the sample can be fragmented prior to primer hybridisation.
  • the hybridisation can be carried out by heating a population of double stranded fragments, thus melting them to be single strands, and allowing the mixture to cool.
  • the sample can be prepared as a single stranded sample without heat denaturing.
  • the nucleic acid fragments in the sample will be single stranded.
  • the fragments can be made single stranded by other chemical treatments, for example exposure to hydroxide.
  • universal adaptors may be attached to the ends.
  • the universal adaptors allow amplification using a single pair of primers complementary to the adaptor.
  • Many methods exist for the preparation of samples of double-stranded DNA, for example for sequencing e.g. Illumina TruSeq and NextEra, 454, NEBnext, Life Technologies etc).
  • the sample may be processed in double stranded form or the sample may be treated (for example using heat denaturation) to give rise to a single stranded form.
  • the double stranded extension products may be treated in order to remove any remaining single stranded primers.
  • the primers may carry modifications such as ribonucleotides in order to facilitate removal of the single stranded primers from the double stranded extension products.
  • the adaptor, or oligonucleotide 5 '-triphosphate adaptor may be single stranded or double stranded.
  • the double stranded adaptor has at least one overhanging single stranded region, and may have two or three overhanging single stranded regions.
  • the overhang serves to act to hybridise to the end of the extended locus primers to which the adaptor is to be attached, and acts as a site which can undergo polymerase extension to make the attached single stranded extended locus primers double stranded.
  • the adaptors can be 'forked' adaptors having regions which are non-complementary as well as regions which are complementary.
  • the attachment of the adaptor can be carried out using a template dependent polymerase. Any polymerase suitable for the incorporation of a nucleotide triphosphate can be used.
  • the adaptor can be thought of as a nucleotide triphosphate attached to an oligonucleotide duplex. Thus the adaptor carries its own template.
  • the adaptor or oligonucleotide 5 '-triphosphate adaptor may have a region of self- complementarity such that the second oligonucleotide may take the form of a hairpin.
  • the hairpin may have 3 '-overhang suitable for polymerase extension.
  • the term single stranded therefore includes a single strand which is in part single stranded, and in part double stranded at certain temperatures, but which can be made single stranded by increasing the temperature.
  • the adaptor may have one or more regions for indexing such that different oligonucleotides can be attached to different samples, thereby allowing sample pooling.
  • the adaptor may have one or more modifications which allow site specific strand cleavage.
  • the adaptor may have one or more uracil bases, thereby allowing site specific cleavage using enzyme treatment.
  • the locus specific primers or the adaptor may be attached to a solid surface, or may contain a modification allowing for subsequent immobilisation or capture.
  • the adaptor attachment may be carried out on a solid support, or the joined products may be captured onto a surface after joining.
  • the locus specific primers or the adaptors may carry a moiety for surface capture, for example a biotin moiety.
  • the attachment may be covalent.
  • the primers may be immobilised on a solid support, and used to capture the single stranded oligonucleotide fragments.
  • the adaptors may be immobilised on a solid support, and used to capture the extended primers.
  • the locus specific primers or the adaptor may be DNA, RNA or a mixture thereof. Where the adaptor contains two strands, one strand may be DNA and one strand may be RNA.
  • An aspect of the invention described herein provides a method of joining two oligonucleotides using a template independent nucleic acid polymerase enzyme such as terminal deoxynucleotidyl transferase (TdT). Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase which catalyses the addition of nucleotides to the 3' terminus of a DNA molecule.
  • TdT terminal deoxynucleotidyl transferase
  • DNTT DNA nucleotidylexotransferase
  • TdT typically adds nucleotide 5 -triphosphates onto the 3 '-hydroxyl of a single stranded first oligonucleotide sequence.
  • the invention as described herein uses a second oligonucleotide carrying a 5 '-triphosphosphate which can be attached to the first oligonucleotide sequence, thus enabling two single stranded oligonucleotides to be joined together, catalysed by TdT.
  • the enzyme can be used to link two oligonucleotide strands, rather than simply adding individual nucleotides.
  • Polyadenylate polymerase is an enzyme involved in the formation of the polyadenylate tail of the 3' end of mRNA.
  • PAP uses adenosine triphosphate (ATP) to add adenosine nucleotides to the 3 ' end of an RNA strand.
  • ATP adenosine triphosphate
  • the enzyme works in a template independent manner.
  • a further aspect of the invention involves the use of PAP to join two oligonucleotides. In the use of PAP, one or more of the oligonucleotides may be RNA rather than DNA.
  • poly(U) polymerase catalyzes the template independent addition of UMP from UTP or AMP from ATP to the 3 ' end of RNA.
  • template independent nucleic acid polymerase enzyme includes any polymerase which acts without requiring a nucleic acid template.
  • template independent nucleic acid polymerase enzyme includes terminal deoxynucleotidyl transferase (TdT), Polyadenylate polymerase (PAP) and poly(U) polymerase (PUP).
  • the template independent nucleic acid polymerase enzyme can be PAP.
  • the template independent nucleic acid polymerase enzyme can be terminal transferase.
  • the oligonucleotides joined can be RNA or DNA, or a combination of both RNA and DNA.
  • the oligonucleotides can contain one or more modified backbone residues, modified sugar residues or modified nucleotide bases.
  • Template independent nucleic acid polymerase enzymes may be sensitive to the bulk of substituents attached to the ribose 3 '- position.
  • the standard substrates for these enzymes are nucleotide triphosphates in which the ribose 3 '- position is a hydroxyl group.
  • the enzyme may be engineered using suitable amino acid substitutions to accommodate any increase in steric bulk.
  • the term template independent nucleic acid polymerase enzyme therefore includes non-naturally occurring (engineered) enzymes.
  • template independent nucleic acid polymerase enzyme includes modified versions of terminal transferase or PAP. Terminal transferase, PUP or PAP may be obtained from commercial sources (e.g. New England Biolabs).
  • the method described herein adds a single stranded oligonucleotide with a 3 ' hydroxyl to a single stranded oligonucleotide with a 5 '-triphosphate moiety.
  • the triphosphate moiety can be attached directly to the 5'-hydroxyl of the second oligonucleotide.
  • the 5 '- oligonucleotide triphosphate can react directly with the 3 '-hydroxyl group of the first oligonucleotide to form a single stranded oligonucleotide containing the first and second sequences linked together via a standard 'natural' phosphomonoester moiety.
  • Such an oligonucleotide can be copied using a polymerase as there are no unnatural linking groups between the first and second oligonucleotides.
  • the use of engineered template independent polymerase enzymes may increase the tolerance for steric bulk at the 3 '-position of the triphosphate nucleotide, and hence allow the use of oligonucleotide strands attached directly to the 3 '-hydroxyl of a nucleotide triphosphate.
  • the triphosphate can be attached through a linker moiety.
  • Linker moieties can be any functionality attached to the terminal 5'hydroxyl of the oligonucleotide strand.
  • the linker moiety can include one or more phosphate groups.
  • the linker may contain a ribose or deoxyribose moiety.
  • the linker may contain one or more further nucleotides.
  • the nucleotides, or the ribose or deoxyribose moieties may be further substituted.
  • the linker may contain a ribose or deoxyribose moiety in which the oligonucleotide is attached to the 2- position of the ribose.
  • the linker may contain a nucleotide in which the remainder of the oligonucleotide is attached via the nucleotide base.
  • the linker may employ one or more carbon, oxygen, nitrogen or phosphorus atoms.
  • the linker acts merely to attach the functional triphosphate moiety to the remainder of the oligonucleotide.
  • the joined oligonucleotides may be copied using a nucleic acid polymerase.
  • the linker should be able to permit a nucleotide polymerase to bridge though the linker in order to copy the strands after joining.
  • the action of the polymerase may be enhanced by using a hybridised primer which can bridge across the linker region.
  • the primer can be designed with a suitable length of sequence to space across the linker region.
  • the sequence can be degenerate/random or simply be a suitable length of known sequence in order to bridge across any gap caused by the linker region.
  • the length of sequence used to bridge the gap can be designed depending on the choice of linker.
  • the sequence can be used as a tag for individual fragments.
  • the tag can be used to assess the level of bias introduced by any amplification reactions. If the tags are say 6 mers of random sequences, there at 4 ⁇ 6 (4096) different variants of different sequence. From a population of fragments from a biological sample, it is highly unlikely that two fragments of the same 'biological' sequence will be joined to a tag with the same 'tag' sequence. Therefore any examples where the fragments and tag are over-represented in the sequencing reaction occur because the particular individual fragment is over-amplified during the PCR reaction when compared to other fragments in the population. Thus the use of 'tags' of variable sequence can be used to help normalise the effects of amplification variability.
  • 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 oligonucleotide with the 5 '-triphosphate may be blocked at the 3 ' end to prevent self joining.
  • the blocking moiety may be a phosphate group or a similar moiety.
  • the 3 ' end may be a dideoxy nucleotide with no 3'-OH group.
  • any blocking group can be removed.
  • the methods can include the step of treatment with a suitable kinase to remove a 3'- phosphate moiety.
  • the kinases may be P K or any suitable kinase.
  • the oligonucleotide with the 5 '-triphosphate may be produced chemically or enzymatically.
  • a suitable nucleotide 5 '-triphosphate may be chemically coupled to a suitable oligonucleotide using suitable chemical couplings.
  • the nucleotide triphosphate may contain an azido (N 3 ) group and the oligonucleotide may contain an alkyne group.
  • a suitable oligonucleotide monophosphate may be turned into a triphosphate either chemically or enzymatically.
  • the sequence of the 5 '-triphosphate 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 adaptor, or oligonucleotide 5 '-triphosphate adaptor may be single stranded or double stranded.
  • the double stranded adaptor has at least one overhanging single stranded region, and may have two or three overhanging single stranded regions.
  • the overhang serves to act to hybridise to the end of the extended locus primers to which the adaptor is to be attached, and acts as a site which can undergo polymerase extension to make the attached single stranded extended locus primers double stranded.
  • the adaptors can be 'forked' adaptors having regions which are non-complementary as well as regions which are complementary.
  • Attachment of the 5 '-triphosphate oligonucleotide may give rise to a join which is not a natural phosphodiester linkage. Such joins may not be substrates for nucleic acid polymerases.
  • the use of 3 '-overhangs, either as hairpins or double stranded adaptors is advantageous as the linking region can be 'bridged' using an oligonucleotide primer sequence which is internal or part of the adaptor.
  • Hybridisation of a primer suitable for extension would also require such an internal spacer, and this lowers the affinity and specificity of the primer hybridisation, whereas no such issues arise where the adaptor has an 'internal' primer which is already hybridised (or in the case of hairpins integral).
  • the attachment of a single 'hairpin' which can be used as both the known end and the extendable primer when preparing a library is therefore advantageous over the attachment of a single known end followed by the hybridisation of a second primer.
  • the pre-formed, or intramolecular hybridisation spans the unnatural join, and allows efficient extension.
  • the oligonucleotide adaptor, or oligonucleotide 5 '-triphosphate adaptor may be single stranded in portions, and have a region of self-complementarity such that the second oligonucleotide may take the form of a hairpin.
  • the adaptor may take the form of a hairpin having a single stranded region and a region of self-complementary double stranded sequence.
  • the hairpin may have 3 '-overhang suitable for polymerase extension. The overhang may stretch across the triphosphate 'linker' region at the 5' end, thus avoiding any issues relating the presence of the 5 '-modification required for TDT incorporation.
  • the self- complementary double stranded portion may be from 5-20 base pairs in length.
  • the overhang may be from 1-10 bases in length.
  • the overhang may contain one or more degenerate bases.
  • the sequence may contain a mixture of bases A, C and T at each position (symbolised as H (not G)). H may be used in cases where the sample is bisulfite treated, and thus does not contain any C bases to which the G would be complementary.
  • the overhang may consist of 1-10 H bases.
  • the overhang may be 2-8 bases, which may be H.
  • the overhang may have a 3 '-phosphate.
  • the overhang may have a 3' -OH.
  • a method of joining a first single stranded oligonucleotide and an at least partly double stranded oligonucleotide adaptor wherein the first single stranded oligonucleotide is a member of a population of fragments obtained by hybridising and extending a locus specific primer complementary to a cleaved biological sample and the second oligonucleotide adaptor has a double stranded portion and a 3 '-overhang which hybridises to the extended locus specific primers, where the joining is carried out between a 3 ' hydroxyl of the extended primer and a 5 '-triphosphate of the adaptor.
  • the adaptor may consist of one or two strands (i.e. a hairpin or a duplex).
  • the attachment may be catalysed by a template dependent or template independent polymerase.
  • Disclosed herein is a method of joining an extended locus specific primer and an oligonucleotide adaptor using a template independent nucleic acid polymerase enzyme, wherein the first single stranded oligonucleotide is a member of a population of fragments obtained by cleaving a biological sample, hybridising a primer thereto and extending the primer and the second oligonucleotide adaptor takes the form of a hairpin having a single stranded region and a region of self-complementary double stranded sequence.
  • the region of self-complementary double stranded sequence is capable of forming a duplex under hybridising conditions.
  • Hybridising conditions may be for example 50 °C in a standard biological buffer as indicated in the experimental section below.
  • the oligonucleotide adaptor may have one or more regions for indexing such that different oligonucleotides can be attached to different samples, thereby allowing sample pooling.
  • the region for indexing may be located as part of the locus primer oligonucleotides.
  • the adaptor oligonucleotide may have one or more modifications which allow site specific strand cleavage.
  • the adaptor oligonucleotide may have one or more uracil bases, thereby allowing site specific cleavage using enzyme treatment with UDG and an endonuclease.
  • Copies of the first locus primer extension products may be produced by extending the 3 '-end of the attached double stranded adaptor or hairpin.
  • the extension of the hairpin produces an extended hairpin.
  • the extended hairpin can also be described as a double stranded nucleic acid having one end joined. Upon denaturation, the extended hairpin becomes a single stranded molecule, but the length of the double stranded portion (for example at least 100 base pairs) means that the sample rapidly hybridises to form the extended hairpin.
  • ligases Alternative methods of attaching adaptors include the use of ligases.
  • the sample may be left double stranded.
  • the sample may be treated to give an overhanging base complementary to an overhang on an adaptor.
  • the blunt ended duplex may be treated to add for example a single 3 ' nucleotide which can then act as an end complementary to an adaptor.
  • the ligation may be blunt ended or cohesive.
  • the method needs to be chosen to avoid the formation of adaptor- adaptor ligation.
  • the ends of the strands are phosphates, and may thus be amenable to adaptors having both 3 ' and 5' phosphates.
  • a hairpin adaptor having a 3 ' phosphate and a 5' phosphate could ligate to the 3 '- hydroxyl of a double stranded nucleic acid, but not the complementary 5' phosphate.
  • the adaptor, having no free hydroxyl groups could not ligate to itself.
  • the desired product can be formed where the extended strand is attached to the adaptor, but the strand derived from the biological sample (having a 5 '-phosphate) remains unligated.
  • the original strand can be denatured, the 3 ' phosphate removed (for example using a kinase such as P K) and the released 3 '-OH can be used in a second extension to copy the extended primer.
  • a double stranded product linked at one end via the adaptor hairpin is produced.
  • 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 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 (HSO 3 ) or metabisulfite ions (S 2 0 5 2 -).
  • bisulfite ions or metabisulfite ions to convert unmethylated cytosines in nucleic acids into uracil is standard in the art and suitable reagents and conditions are well known.
  • a population of DNA strands having one or more abasic sites may be produced by subjecting a population of nucleic acid molecules to acid hydrolysis.
  • the population may be subjected to acid hydrolysis by incubation at an acidic pH (for example, pH 5) and elevated temperature (for example, greater than 70 °C).
  • acidic pH for example, pH 5
  • elevated temperature for example, greater than 70 °C
  • a proportion of the purine bases in the nucleic acid strands will be lost, to generate abasic sites.
  • the number of abasic sites formed depends on the pH, concentration of buffer, temperature and length of incubation.
  • a population of DNA strands having one or more abasic sites may be produced by treating the population of nucleic acid strands with uracil-DNA glycosylase (UDG).
  • the population may be treated with UDG by incubation with UDG at 37°C.
  • UDG excises uracil residues in the nucleic acid strands leaving abasic sites.
  • UDG may be obtained from commercial sources.
  • 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.
  • Methods of the invention may be useful in producing populations of mono-adapted single stranded nucleic acid fragments i.e. nucleic acid strands having an adaptor oligonucleotide attached to their 3 ' termini.
  • populations of 3 ' adapted single stranded nucleic acid fragments may be used directly for sequencing and/or amplification.
  • the sequence of the adaptor oligonucleotide may be entirely known, or may include a variable region.
  • the sequence may a universal sequence such that each joined sequence has a common 'adaptor' sequence attached to one end.
  • the attachment of an adaptor to one end of a pool of fragments of variable sequence means that copies of the variable sequences can be produced using a single 'extension' primer.
  • the extended locus sequences may be determined by hybridising the fragments onto a solid support carrying an array of primers complementary to the adaptor oligonucleotide sequence.
  • the joined fragments may have an adaptor modification at the 3'- end which allows attachment to a solid support.
  • the methods disclosed may further include the step of producing one or more copies of the primer extended 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 oligonucleotide adaptor 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 adaptor is a hairpin
  • the internal 3'-OH of the hairpin may be used, and the double stranded polynucleotide is in the form of an extended hairpin.
  • the hairpin can contain a cleavage site (for example a uracil nucleotide). Upon cleavage, the hairpin becomes two strands rather than one, and is no longer a hairpin.
  • a cleavage site for example a uracil nucleotide.
  • the hairpin becomes two strands rather than one, and is no longer a hairpin.
  • the sample becomes a known end (from the universal region of the locus primer), and unknown extension region from the sample of interest (the sequence of which can be determined) and a known end from the attached adaptor.
  • the double stranded polynucleotides can be amplified using primers complementary to both known 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. Amplification of circular fragments can be carried out using primers complementary to two regions of the single adaptor sequence.
  • a second adaptor may be attached to a product after a second extension.
  • the second adaptor may comprise a self-complementary double stranded region (i.e. a hairpin).
  • kits and components for carrying out the invention.
  • a kit for use in preparing a nucleic acid sample comprising a polynucleotide having a triphosphate moiety at the 5 '-end and population of locus specific primers.
  • the kit may contain a nucleotide 5 -triphosphate adaptor having any of the features described herein.
  • the two or more different sequences may include a fixed sequence capable of hybridising to an extension primer, and a variable sequence which acts as a tag to identify the adaptor (and hence the identify of the sample to which the adaptors are attached).
  • each member of the population contains a common universal sequence and one of a plurality of locus- specific regions wherein each locus specific region contains only the nucleic acid bases A, C and T such that the locus is complementary to a sample treated with bisulfite.
  • Such primers may be included in a kit with a polynucleotide having a triphosphate moiety at the 5'-end.
  • the primers may comprise 50 or more locus specific sequences in the population.
  • the adapter in the kits may be single stranded or double stranded.
  • the adapter in the kits may be a hairpin.
  • a kit for use in selecting fragments from a nucleic acid sample comprising a plurality of locus specific primers, a hairpin polynucleotide having a triphosphate moiety at the 5 '-end and a terminal transferase or polymerase.
  • the population of locus specific nucleic acid primers may be such that each member of the population contains a common universal sequence and one of a plurality of locus-specific regions wherein each locus specific region contains only the nucleic acid bases A, C and T such that the locus is complementary to a sample treated with bisulfite.
  • Figure 1 shows the method of the invention where the primers contain a universal region.
  • the primers are subject to multiple cycles of hybridisation and extension.
  • An adaptor is attached shown in the form of a hairpin.
  • the 3 ' OH of the hairpin is used for extension.
  • the hairpin is cleaved (also cleaving the first extended strand), and the resulting products are sequenced. This method was used to generate the data shown below.
  • Figure 2 shows the method where the locus primers do not have a universal region.
  • the primers are subject to multiple cycles of hybridisation and extension.
  • An adaptor is attached shown in the form of a hairpin.
  • the 3' OH of the hairpin is used for extension.
  • a second adaptor is attached (using a ligase as shown).
  • the hairpin is cleaved (also cleaving the first extended strand), and the resulting products are sequenced.
  • Figure 3 shows a 2% agarose gel of Loci Targeting libraries obtained from bisulfite converted Phi X genomic DNA.
  • Figure 4 shows Graphic representation (SeqMonk) of the sequencing data obtained from bisulfite converted Phi X multiplex loci targeting libraries.
  • Disclosed herein is a method of joining a first single stranded oligonucleotide and an oligonucleotide adaptor using a template independent nucleic acid polymerase enzyme, wherein the first single stranded oligonucleotide is obtained by extending a primer and the second oligonucleotide adaptor takes the form of a hairpin having a single stranded region and a region of self-complementary double stranded sequence.
  • sequenced single stranded products represent a sub-set of the original sample selected by the design of the primers.
  • Disclosed herein is a method of selecting and analysing a subset of a nucleic acid sample comprising; a) providing a sample containing a population of nucleic acid molecules, b) treating the population with bisulfite to produce a sample of nucleic acid strands containing a mixture of first single stranded oligonucleotides of different sequence,
  • Suitable sequencing methods are well known in the art, and include Ulumina sequencing, pyrosequencing (for example 454 sequencing) or Ion Torrent sequencing from Life TechnologiesTM).
  • Populations of nucleic acid molecules with a 3 ' adaptor oligonucleotide and a 5' 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 are not found within the human genome, or other sample genome of interest.
  • the nucleic acid strands in the population to be sequenced 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.
  • 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. a 20-30 nucleotides); a sequence that hybridises to sequencing primer (e.g. a 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 adaptor may be for example 5-20 bases of a first complementary sequence, a single stranded loop comprising a sequence that hybridises to the solid support and the sequencing primer (e.g.
  • the hairpin constructs may be 60 to 100 nucleotides or more in length.
  • 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 or to the universal sequence on the locus specific primers.
  • 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 or the universal sequence on the locus specific primers.
  • 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.
  • Phi X genomic DNA (Phi X 174 NEB, 500 ng) was bisulfite converted using the TrueMethyl conversion kit (CEGX) following the manufacturers specification. The DNA was then quantified by Qubit ssDNA assay kit.
  • Step 3 Probe hybridization
  • Hybridised probes were snap cooled at 4°C and extended at 37°C for 30 min using 2.5 U of the Klenow Fragment (3 '-5' exo- Enzymatics) and 10 mmol dNTP mix, in the same buffer conditions of step 3.
  • the products obtained were purified with 2x 18% PEG Ampure XP Beads (18% PEG-8000, 1 M NaCl, 1 mM EDTA, 10 mM Tris pH 8) according to manufacturers instructions but using 80:20 freshly prepared acetonitrile:water instead of 80:20 ethanol :water. Samples were eluted from the beads in 10 ⁇ ultra pure water.
  • the hairpin triphosphate adaptor was prepared as follows: a hairpin oligo (1250 ⁇ , Biomers GMBH) with a 3' DBCO modification was reacted with an azido-3'- deoxyadenosine-5 '-triphosphate (1000 ⁇ , JenaBiosciences) for 2h at 10°C in 10 mM Tris- HC1 pH 7. The final sequence is listed in Table 2 below. An aliquot of this hairpin adaptor (40 pmol) was then used to tag the end of the Klenow-extended molecules as follows.
  • a mix of the hairpin adaptor and purified fragments (1.2 pmol) from step 2 were denaturated at 95°C for 3 min in lx TdT buffer (lOOmM Tris-acetate, 1.25 mM CoAc2, 125 ⁇ g/mL BSA, pH 6.6 @ 25 °C) and then briefly snap cooled.
  • TdT Enzymatics (20 U) was then added to catalyse the hairpin adaptor incorporation (37°C for 30 min). Reaction was stopped with 20 mM EDTA and TdT denaturated at 95°C for 5 min.
  • the products obtained were purified with 2x Ampure XP Beads resuspended in a 30% PEG solution (30% PEG-8000, 1 M NaCl, 1 mM EDTA, 10 mM Tris pH 8) according to manufacturers instructions but using 80:20 freshly prepared acetonitrile:water instead of 80:20 ethanol: water. Samples were eluted from the beads 10 ⁇ ultra pure water
  • the 10 ⁇ adapted DNA eluted from step 7 was mixed with the P K/Klenow cocktail (5U PNK, 2.5 U Klenow exo-, 10 mmol dNTP mix in IX Blue buffer) to dephosphorylate the 3' end of the attached hairpin adaptor and to synthesise the full length, complementary bottom strand by extension from the annealed hairpin adaptor using Klenow.
  • P K/Klenow cocktail 5U PNK, 2.5 U Klenow exo-, 10 mmol dNTP mix in IX Blue buffer
  • Extended fragments were digested adding a UDG/EndoVIII mix (1 U of UDG and 5 U of Endo VIII) directly to the reaction step 8. Products were purified using magnetic beads as described in step 7 and eluted from the beads 10 ⁇ ultra pure water.
  • Step 10 PCR amplification
  • PCR amplification was performed on Agilent Surecycler 8800 thermocycler using 1 U of VeraSeq Ultra DNA polymerases (Enzymatics). Thermocycling conditions were 35 cycles of: Denaturation at 95°C for 30 sec
  • the primers used included a sequence that hybridizes to an Illumina flow cell and a specific index tag (represented by a string of 6N nucleotides). PCR products were purified as described in step 5 and eluted in 20 ⁇ ultra pure water. Figure 3 shows the 2% non- denaturing agarose gel of the 6 purified libraries prepared in duplicate.
  • Step 1 Sequencing and analysis:
  • Sequencing was carried out on an Illumina Miseq sequencer with a paired end run (2x 75 bp).
  • the 6 libraries were pooled at 2 nM and then diluted to 20 pM before loading to the flow cell and sequenced, according to the manufacturers instructions.
  • the raw output fastq read sequences were quality filtered and trimmed to remove the adaptor sequences with the software Trim Galore.
  • the data was aligned to the Phi X genome with Bismark software and visualized by SeqMonk.
  • the picture in Figure 4 shows the visualization by SeqMonk of the sequencing data obtained from a multiplex loci targeting library. Six distinct peaks can be observed corresponding to read pile-ups at the expected target regions. A more detailed analysis of the sequence reads showed that the starting position (see Table 1) was correct for each region. Sequence reads from the target regions were more highly represented compared to not-specific sequences illustrative of a successful enrichment for the target regions.

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Abstract

La présente invention concerne la préparation d'échantillons d'acides nucléiques pour l'analyse et certains procédés et outils pour la sélection de régions spécifiques d'intérêt d'un échantillon d'acide nucléique.
PCT/GB2016/051083 2015-04-20 2016-04-20 Enrichissement d'échantillons d'acide nucléique WO2016170319A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9822394B2 (en) 2014-02-24 2017-11-21 Cambridge Epigenetix Limited Nucleic acid sample preparation
US10323269B2 (en) 2008-09-26 2019-06-18 The Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10428381B2 (en) 2011-07-29 2019-10-01 Cambridge Epigenetix Limited Methods for detection of nucleotide modification
US10563248B2 (en) 2012-11-30 2020-02-18 Cambridge Epigenetix Limited Oxidizing agent for modified nucleotides
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US12024750B2 (en) 2018-04-02 2024-07-02 Grail, Llc Methylation markers and targeted methylation probe panel

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030211494A1 (en) * 2002-05-03 2003-11-13 Prokaria, Ltd. Retrieval of genes and gene fragments from complex samples
US20040038215A1 (en) * 2002-08-23 2004-02-26 Shiv Kumar Oligonucleotide tagged nucleoside triphosphates (OTNTPs) for genetic analysis
US20040110153A1 (en) * 2002-12-10 2004-06-10 Affymetrix, Inc. Compleixity management of genomic DNA by semi-specific amplification
US20040137439A1 (en) * 2003-01-10 2004-07-15 Linden Technologies, Inc. Nucleic acid amplification
US20110294129A1 (en) * 2009-01-05 2011-12-01 Biomerieux Method for amplifying and/or detecting nucleic acids, kits and uses of said method
CN103571822A (zh) * 2012-07-20 2014-02-12 中国科学院植物研究所 一种用于新一代测序分析的多重目的dna片段富集方法
WO2014199336A2 (fr) * 2013-06-13 2014-12-18 Vela Operations Pte.Ltd. Flux de production de ngs amélioré
WO2015124955A1 (fr) * 2014-02-24 2015-08-27 Cambridge Epigenetix Limited Préparation d'échantillons d'acides nucléiques

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030211494A1 (en) * 2002-05-03 2003-11-13 Prokaria, Ltd. Retrieval of genes and gene fragments from complex samples
US20040038215A1 (en) * 2002-08-23 2004-02-26 Shiv Kumar Oligonucleotide tagged nucleoside triphosphates (OTNTPs) for genetic analysis
US20040110153A1 (en) * 2002-12-10 2004-06-10 Affymetrix, Inc. Compleixity management of genomic DNA by semi-specific amplification
US20040137439A1 (en) * 2003-01-10 2004-07-15 Linden Technologies, Inc. Nucleic acid amplification
US20110294129A1 (en) * 2009-01-05 2011-12-01 Biomerieux Method for amplifying and/or detecting nucleic acids, kits and uses of said method
CN103571822A (zh) * 2012-07-20 2014-02-12 中国科学院植物研究所 一种用于新一代测序分析的多重目的dna片段富集方法
WO2014199336A2 (fr) * 2013-06-13 2014-12-18 Vela Operations Pte.Ltd. Flux de production de ngs amélioré
WO2015124955A1 (fr) * 2014-02-24 2015-08-27 Cambridge Epigenetix Limited Préparation d'échantillons d'acides nucléiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "TruSeq DNA Methylation Library Preparation Guide", 1 December 2014 (2014-12-01), pages 1 - 20, XP055283756, Retrieved from the Internet <URL:http://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_truseq/truseq-dna-methylation/truseq-dna-methylation-library-prep-guide-15066014-a.pdf> [retrieved on 20160627] *
ANONYMOUS: "Whole-Genome bisulfite sequencing", APPLICATION NOTE: EPIGENETICS, 30 July 2014 (2014-07-30), pages 1 - 2, XP055283449, Retrieved from the Internet <URL:http://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/appnote-methylseq-wgbs.pdf> [retrieved on 20160624], DOI: 10.1093/bioinformatics/btu170 *
ERIC J DEVOR ET AL: "DNA Oligonucleotides with Cleavable 3'-terminal Ribonucleotides: A Versatile Molecular Tool", 1 January 2005 (2005-01-01), XP055283827, Retrieved from the Internet <URL:https://intranet.pasteur.edu.uy/publico/bonilla/Protocolos/pcr/DNA_Oligonucleotides_with_Cleavable_3_Prime_Termini.pdf> [retrieved on 20160627] *
LIU CHIH LONG ET AL: "Development and validation of a T7 based linear amplification for genomic DNA", BMC GENOMICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 4, no. 1, 9 May 2003 (2003-05-09), pages 19, XP021014452, ISSN: 1471-2164, DOI: 10.1186/1471-2164-4-19 *

Cited By (22)

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US10731204B2 (en) 2008-09-26 2020-08-04 Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10337053B2 (en) 2008-09-26 2019-07-02 Children's Medical Center Corporation Labeling hydroxymethylated residues
US10767216B2 (en) 2008-09-26 2020-09-08 The Children's Medical Center Corporation Methods for distinguishing 5-hydroxymethylcytosine from 5-methylcytosine
US12018320B2 (en) 2008-09-26 2024-06-25 The Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10443091B2 (en) 2008-09-26 2019-10-15 Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10465234B2 (en) 2008-09-26 2019-11-05 Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10508301B2 (en) 2008-09-26 2019-12-17 Children's Medical Center Corporation Detection of 5-hydroxymethylcytosine by glycosylation
US10533213B2 (en) 2008-09-26 2020-01-14 Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10774373B2 (en) 2008-09-26 2020-09-15 Children's Medical Center Corporation Compositions comprising glucosylated hydroxymethylated bases
US10612076B2 (en) 2008-09-26 2020-04-07 The Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US11208683B2 (en) 2008-09-26 2021-12-28 The Children's Medical Center Corporation Methods of epigenetic analysis
US10323269B2 (en) 2008-09-26 2019-06-18 The Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US11072818B2 (en) 2008-09-26 2021-07-27 The Children's Medical Center Corporation Selective oxidation of 5-methylcytosine by TET-family proteins
US10793899B2 (en) 2008-09-26 2020-10-06 Children's Medical Center Corporation Methods for identifying hydroxylated bases
US10428381B2 (en) 2011-07-29 2019-10-01 Cambridge Epigenetix Limited Methods for detection of nucleotide modification
US10563248B2 (en) 2012-11-30 2020-02-18 Cambridge Epigenetix Limited Oxidizing agent for modified nucleotides
US9822394B2 (en) 2014-02-24 2017-11-21 Cambridge Epigenetix Limited Nucleic acid sample preparation
US12024750B2 (en) 2018-04-02 2024-07-02 Grail, Llc Methylation markers and targeted methylation probe panel
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US11685958B2 (en) 2018-09-27 2023-06-27 Grail, Llc Methylation markers and targeted methylation probe panel
US11725251B2 (en) 2018-09-27 2023-08-15 Grail, Llc Methylation markers and targeted methylation probe panel
US11795513B2 (en) 2018-09-27 2023-10-24 Grail, Llc Methylation markers and targeted methylation probe panel

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