WO2015145133A1 - Procédé de préparation d'acides nucléiques - Google Patents

Procédé de préparation d'acides nucléiques Download PDF

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WO2015145133A1
WO2015145133A1 PCT/GB2015/050871 GB2015050871W WO2015145133A1 WO 2015145133 A1 WO2015145133 A1 WO 2015145133A1 GB 2015050871 W GB2015050871 W GB 2015050871W WO 2015145133 A1 WO2015145133 A1 WO 2015145133A1
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nucleic acids
adaptor
nucleic acid
double
population
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PCT/GB2015/050871
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English (en)
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Shankar Balasubramanian
Eun-Ang RAIBER
Gordon MCINROY
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Cambridge Enterprise Limited
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Priority to EP15713006.3A priority Critical patent/EP3122879A1/fr
Publication of WO2015145133A1 publication Critical patent/WO2015145133A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present inventors have developed a process that provides improved yields of nucleic acids, for example bisulfite-treated nucleic acids, that carry adaptors on both ends and are suitable for sequencing .
  • fragments and the associated information can be recovered by employing a two-step ligation procedure, where a first adapter is added before bisulfite treatment and a second adapter afterwards .
  • An aspect of the invention provides a method of preparing a nucleic acid library comprising;
  • Each double-stranded nucleic acid in the library may comprise a target nucleic acid from the population with the first adaptor at a first end and the second adaptor at a second end.
  • the nucleic acids with the 3' adapto equence may be treated with bisulfite.
  • the double-stranded nucleic acids in the library may be denatured to produce a library of nucleic acid strands having a first adaptor sequence at a first end and a second adaptor sequence at a second end.
  • Each nucleic acid strand in the library may comprise the sequence of a target nucleic acid from the population with the first adaptor sequence at a first end and the second adaptor sequence at a second end.
  • nucleic acids in the nucleic acid library may be interrogated, for example to determine the identity of one or more bases in the target nucleic acid sequence.
  • Suitable methods of interrogation include sequencing or hybridisation, for example to a probe, e.g. a probe immobilised on an array.
  • a method may comprise sequencing nucleic acids in the nucleic acid library following preparation as described above.
  • a nucleic acid library is a diverse collection of single or double stranded target nucleic acids.
  • the nucleic acids have adapted ends i.e. the nucleic acids in the library have an adaptor at each end.
  • the presence of terminal adaptors at the ends of the target nucleic acid sequence allows the nucleic acids in the library to be sequenced.
  • all or substantially all of the nucleic acids in a library are sequenceable (i.e. the nucleic acids in the library each comprise adaptors at both ends) following production as described herein.
  • the population of target nucleic acids may be obtained or isolated from a sample of cells, for example, mammalian cells, preferably human cells. Suitable samples include isolated cells and tissue samples, such as biopsies.
  • target nucleic acids may be obtained or isolated from neural cells, including neurons and glial cells, contractile muscle cells, smooth muscle cells, liver cells, hormone synthesising cells, sebaceous cells, pancreatic islet cells, adrenal cortex cells, fibroblasts, keratinocytes , endothelial and urothelial cells, osteocytes, and chondrocytes.
  • neural cells including neurons and glial cells, contractile muscle cells, smooth muscle cells, liver cells, hormone synthesising cells, sebaceous cells, pancreatic islet cells, adrenal cortex cells, fibroblasts, keratinocytes , endothelial and urothelial cells, osteocytes, and chondrocytes.
  • genomics The analysis of nucleic acids from a single cell may, for example, allow genetic variability and epigenetic variability in individual cells and cell-types to be determined.
  • the population of target nucleic acids may obtained from a sample of biological fluid, for example a sample amniotic fluid, cerebrospinal fluid, mucus , sebum, blood, plasma, serum, urine or saliva
  • a sample of biological fluid for example a sample amniotic fluid, cerebrospinal fluid, mucus , sebum, blood, plasma, serum, urine or saliva
  • genomic DNA or RNA Methods of extracting and isolating target nucleic acids, such as genomic DNA or RNA, from an individual cell, a sample of cells or a biological fluid, are well-known in the art.
  • genomic DNA or RNA may be isolated using any convenient isolation
  • fragmented nucleic acid sample may be used as described herein.
  • nucleic acid fragments may be repaired to produce a population of blunt-ended nucleic acids.
  • Suitable methods of repairing nucleic acid ends are well-known in the art.
  • fragmented nucleic acids may be converted into blunt-ended molecules by filling in 5' overhangs using a 5' ⁇ 3' polymerase, and removing 3' overhangs using a 3' to 5' exonuclease, in accordance with standard techniques .
  • Suitable polymerases include T4 DNA polymerase and/or Klenow fragment.
  • the ends of the nucleic acids are not treated with a 5' kinase, such as T4 polynucleotide, and remain unphosphorylated at the 5' ends. This may be useful in preventing the ligation of adaptors or other nucleotide sequences to the 5' ends of the nucleic acid strands .
  • the target nucleic acids in the population are blunt-ended and may, in some preferred
  • embodiments comprise free hydroxyls at both the 5' and 3' ends of each strand.
  • Suitable techniques and kits for the end-repair of nucleic acid molecules are widely available from commercial suppliers (e.g. End- ItTM, Epicentre; NEBNextTM end repair Module, New England Biolabs; Fast DNA End Repair, Thermo Fisher Scientific; DNA End Repair Mix, Life Technologies; Paired-End Sample Prep Kit, Illumina Inc) .
  • a suitable adaptor for a single-stranded nucleic acid may comprise an adaptor sequence .
  • a suitable adaptor for a double stranded nucleic acid may comprise an adaptor sequence and a complementary sequence which hybridises to all or part of the adaptor sequence, such that the adaptor comprises a double-stranded portion that is ligated to the target nucleic acid and a single-stranded overhang (i.e. a double-stranded region proximal to the target nucleic acid and a single-stranded tail).
  • a nucleic acid in a library may comprise a first adaptor at one end (e.g. a first end) and a second adaptor at the other end (e.g. a second end) .
  • the sequence of the target nucleic acid is located between the first and second adaptors .
  • the nucleic acids in the library may have the same first adaptor at their 3' ends and the same second adaptor at their 5' ends i.e. all of the nucleic acids in the library may be flanked by the same pair of adaptors.
  • the first and second adaptors may be different or more preferably the same (i.e. the nucleic acids may have the same adaptor at each end) .
  • all of the nucleic acids in a library may comprise the same adaptor sequence, or adaptor sequences which differ only in an index sequence.
  • the adaptors and adaptor sequences are synthetic sequences that are not found within the mammalian genome.
  • Adaptors suitable for use in sequencing nucleic acids are well-known in the art. Adaptors are generally specific for a sequencing platform and the sequence of the adaptor therefore depends on the specific sequencing method to be employed. Adaptors suitable for any specific sequencing method are well-known in the art and may be designed and produced using known techniques or obtained from commercial sources . The choice of adaptor nucleotide sequence depends on the sequencing method employed and suitable adaptors . Suitable sequencing platforms include Sanger sequencing, Solexa- Illumina sequencing platforms, such as HiseqTM, MiSeqTM and NextSeqTM, semiconductor array sequencing ( IonTorrentTM; LifeTech) ,
  • pyrosequencing e.g. 454 Sequencing; Roche 454
  • SMRTTM single molecule real-time sequencing
  • adaptors suitable for any of these sequencing platforms may be used in the methods described herein.
  • adaptors may include a region that is complementary to the universal primers on a 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 sequence and extended allows the sequence of the nucleic acid molecule to be read) .
  • Adaptor sequences suitable for use as described herein may consist of 20 to 80 nucleotides long.
  • the adaptor may comprise a sequence that hybridises to complementary primers immobilised on the solid support (e.g. 20-30 nucleotides); a sequence that hybridises to a sequencing primer (e.g. 30-40
  • a suitable adaptor may be 56-80
  • one or more of the adaptors may have all the cytosine bases in the methylated form. If the adaptors contain unmethylated cytosines, the adaptors are altered during the bisulfite conversion such that any unmethylated cytosines become uracil. Thus any adaptors attached to the sample prior to bisulfite exposure may be free of unmethylated cytosine bases .
  • the adaptor sequence comprises 5 ' methyl-cytosines instead of cytosines, in order to prevent deamination of cytosines in the bisulfite conversion reaction. Preventing the conversion of cytosines in the adaptor sequence to U (read as T) may be useful in ensuring that the adaptor sequence is able to hybridise to the flowcell of the sequencing platform.
  • the 3' adaptor sequence may comprise one or more modified nucleotides or nucleotide analogues that are resistant to bisulfite damage.
  • the 3' adaptor sequence consists of modified nucleotides or nucleotide analogues or
  • oligomers such as 2- fluorocytosine, 2 aza-cytosine and 2-O-methylcytosine, 6-substituted nucleotides, sua as 6-fluorocytosine, 6-O-methylcytosine 6-aza- cytosine and/or othe modified nucleotides.
  • 2- substituted nucleotides such as 2- fluorocytosine, 2 aza-cytosine and 2-O-methylcytosine
  • 6-substituted nucleotides sua as 6-fluorocytosine, 6-O-methylcytosine 6-aza- cytosine and/or othe modified nucleotides.
  • Suitable modified nucleotides prevent the bisulfite from forming an adduct with the bases, reducing the propensity for abasic site formation and hence reducing the chance of fragmentation.
  • An adaptor may comprise or consist of nucleotide sequences that are common to the members of the library i.e. each nucleic acid in the library may contain the same adaptor sequences . Libraries produced from different sources may be mixed before sequencing.
  • one or both of the adaptors attached to a target nucleic acid may comprise an individual barcode or index nucleotide sequence that identifies the source of the nucleic acid (e.g. the sample) and allows the multiple samples to be sequenced in a multiplex sequencing reaction. Outside the index, the sequences of the adaptors or adaptor sequences may be the same for all the nucleic acids in the library.
  • the nucleotide sequence of the index allows unambiguous
  • a suitable index for the multiplex sequencing of 24 samples may consist of at least 6 nucleotides, preferably 6 nucleotides (Craig DW et al. 2008. Nat Methods 5, 887; Cronn R et al . 2008. Nucleic Acids Res, 36, el22) .
  • the use of indexes, barcodes or identifiers in sequencing reactions is well-known in the art.
  • the adaptors at the 3' and 5' ends of the nucleic acids in a library produced as described herein from a first sample may have the same "core" sequence as the sequences at the 3' and 5' ends of nucleic acids in a library produced from a second sample except for the index, which is unique to the nucleic acid strands from a particular sample.
  • an index of n/4 bases may differ in the sequences at the 3' and/or 5' ends of nucleic acids from different populations. This maintains the specificity of the adaptor sequences for the sequencing platform (e.g.
  • the adaptor oligonucleotide may be ligated to the 3' ends by any convenient ligation method.
  • the adaptor oligonucleotide may be ligated to the 3' ends without ligation or other modification of the 5' ends of the nucleic acids.
  • the adaptor oligonucleotide may be linked to binding tag, for example via a cleavable linker. This is described in more detail below.
  • the adaptor oligonucleotide may be added to the 3' ends of the double-stranded target nucleic acids by any convenient method.
  • the adaptor oligonucleotide may be attached to the 3' ends by suitable ligation methods, including double-stranded ligation, single-stranded ligation, blunt-ended ligation or overhanging ligation .
  • the adaptor oligonucleotide is added to the 3' ends of the double-stranded target nucleic acids as part of an at least partially double-stranded complex or molecule.
  • ligation may be carried out using an enzyme with double-stranded ligase activity in the presence of an inert hybridisation partner that hybridises to the adaptor oligonucleotide but does not ligate to the nucleic acids .
  • oligonucleotide may be non-ligatable through the absence of an OH group, for example due to the presence of a blocking group, such as a halogen, or more preferably a dideoxynucleotide .
  • a blocking group such as a halogen, or more preferably a dideoxynucleotide .
  • oligonucleotide specifically to the 3' ends of the nucleic acids may be facilitated by the modification of the nucleic acid ends .
  • a 3' overhanging adenine (A) residue may be present at the ends of the nucleic acids.
  • the ligation complex may comprise a 3' overhanging T residue which facilitates ligation to nucleic acids in the population comprising a 3' overhanging A residue.
  • the adaptor sequence may be ligated to the 3' ends of the double-stranded target nucleic acids and the
  • the adaptor sequence of the double-stranded adaptor may be linked to a binding tag, for example via a cleavable linker. This is described in more detail below.
  • the double stranded adaptor comprises a cleavage site at the 3' end of the complementary sequence.
  • the adapted nucleic acids may be cleaved at the cleavage site to remove the complementary sequence ligated to the 5' ends of the nucleic acids, such that adapted nucleic acids have an adaptor sequence at the 3' end, but lack additional sequence at the 5' end.
  • a double-stranded adaptor may comprise more than one cleavage site, for example two or three.
  • the double-stranded adaptor may comprise cleavage site at the 3' end of the complementary sequence and one or more additional cleavage sites, for example within a hairpin sequence or elsewhere.
  • the double-stranded adaptor is a hairpin adaptor which comprises a hairpin nucleotide sequence that links the adaptor sequence and the complementary sequence i.e. the double-stranded adaptor consists of a polynucleotide chain which forms a double stranded region and a single-stranded hairpin region. This may be useful in protecting the ends of the nucleic acids from damage, for example during bisulfite treatment.
  • the hairpin adaptor comprises a first cleavage site at the 3' end of the complementary sequence and a second cleavage site at the 5' end of the adaptor sequence. Cleavage of the first and second cleavage sites produces a population of nucleic acids having the adaptor sequence at the 3' ends but lacking an adaptor sequence at the 5' ends.
  • Suitable cleavage sites include any site that is specifically cleavable by enzymatic, chemical or other means.
  • Suitable cleavag sites are well known in the art and include modified nucleotides, such as 8-oxoguanine or 8-oxoadenine , which are cleavable by formamidopyrimidine [fapy]-DNA glycosylase (Fpg) and restriction enonuclease recognition sites.
  • the nucleic acids may be treated with bisulfite following addition of the adaptor sequence to the 3' ends.
  • bisulfite may have unmodified 5' ends.
  • a double stranded adaptor such as a hairpin adaptor
  • the nucleic acids treated with bisulfite may have complementary sequences ligated to the 5' ends. These complementary sequences may be removed after the bisulfite treatment to produce bisulfite treated nucleic acids having an adaptor sequence at the 3' end but not the 5' end.
  • the adaptor sequence may be added to the 3' ends of the nucleic acids by ligating a double-stranded adaptor comprising the adaptor sequence hybridised to a complementary sequence to the ends of the nucleic acids, preferably a hairpin adaptor, such that the adaptor sequence is ligated to the 3' ends of the double- stranded nucleic acids and the complementary sequence is ligated to the 5' ends of the double-stranded nucleic acids. After treatment with bisulfite, the 5' ends of the nucleic acids are cleaved to remove the complementary sequence.
  • a method of preparing a nucleic acid library may comprise;
  • the double-stranded adaptor is a hairpin adaptor comprising a single stranded hairpin nucleotide sequence that links the hybridised adaptor and complementary sequences, as described above .
  • the population of double-stranded nucleic acids may then be treated in accordance with steps (i) to (vi) above.
  • the nucleic acids may be subjected to an additional treatment before treatment with
  • bisulfite This may be useful, for example, in performing variants of standard bisulfite sequencing methods (BS-seq) , for example to identify specific cytosine modifications, such as 5hmC, 5fC and 5caC.
  • BS-seq standard bisulfite sequencing methods
  • Methods may comprise treating the nucleic acids with a reducing agent, and then treating the reduced nucleic acids with bisulfite.
  • Suitable reducing agents are well-known in the art and include NaBH 4 , NaCNBH 4 and LiBH 4 .
  • Techniques for reductive bisulfite sequencing are available in the art (redBS-seq; WO2013/017853 ) .
  • Methods may comprise treating the nucleic acids with 3- glu.cosyltran.sfera.se in the presence of UDP-Glucose to add a glucosyl protecting group to 5hmC residues in the nucleic acids; treating the nucleic acids with TET to oxidise 5mC residues in the nucleic acids to 5caC and then treating the TET-oxidised nucleic acids with bisulfite.
  • Techniques for TET-assisted bisulfite sequencing are well-known in the art (TAB-seq; Yu et al (2012) Nat Protoc. 7 (12) 2159-2170; Yu et al Cell (2012) 149(6) : 1368-1380) and reagents are available from commercial sources (e.g, Wisegene LLC USA) .
  • Methods may comprise labelling 5caC residues in the nucleic acids with l-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride
  • Methods may comprise labelling 5fC residues in the nucleic acids with O-ethylhydroxylamine; and then treating the labelled nucleic acids with bisulfite.
  • Techniques for 5fC chemical modification- assisted bisulfite sequencing are well-known in the art (fCAB-seq; Song et al (2013) Cell 153 1-14) .
  • the strands of nucleic acids in the libraries described herein may comprise nucleotide sequences that are bisulfite-treated (i.e.
  • bisulfite-treated sequences i.e. containing adenine instead of unmodified cytosine in the untreated sequence
  • nucleotide sequences that are the complement of the sequences complementary to bisulfite-treated sequences i.e. containing thymine instead of unmodified cytosine in the untreated sequence
  • unmodified cyotosines may be replaced by uracil in nucleic acid strands following bisulfite treatment.
  • the resultant double-stranded molecules comprise a uracil-containing strand and a non-uracil containing complementary strand. Either or both of these strands may be subsequently isolated and sequenced.
  • sequences of bisulfite-treated nucleic acids may be useful in determining the presence or frequency of modified cytosine residues, such as 5mC, in samples of nucleic acid.
  • Bisulfite treatment causes extensive depyrimidination and strand cleavage in populations of nucleic acids. For example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the nucleic acids may be cleaved during bisulfite treatment.
  • the nucleic acids in the populations used to produce libraries as described herein may include nucleic acids that are not cleaved by the bisulfite treatment and nucleic acids that are cleaved by the bisulfite treatment.
  • the bisulfite treated population therefore comprises nucleic acid strands of a range of sizes, depending on whether cleavage has occurred and its location relative to the 3' adaptor sequence.
  • a library produced as described herein may comprise nucleic acids ranging from lObp to 5kb, 20bp to 2kb or 30bp to lkb (Ehrich et al Nucleic Acids Research, 2007, Vol. 35, No. 5 e29) .
  • the libraries contain a greater proportion of the initial nucleic acid population than libraries that only comprise uncleaved nucleic acids.
  • the number of nucleic acids in the nucleic acid library may be greater than 0.1%, greater than 1%, greater than 5% or greater than 10% of the number of nucleic acids in the initial population.
  • Libraries produced by the methods described herein may contain sufficient sequenceable nucleic acid molecules to allow sequencing without amplification i.e. the nucleic acids in the library may be sequenced without being amplified.
  • a nucleic acid library is produced as described herein without any amplification of the nucleic acids in the sample.
  • Bisulfite treatment as described above may denature double-strande 3' adapted nucleic acids to produce nucleic acid strands of a rang of sizes that all have the adaptor sequence at the 3' end.
  • double-stranded nucleic acids may be denatured following addition of the 3' adaptor sequence without bisulfite treatment .
  • the nucleic acids may be denatured to disrupt any inter or intra molecular hybridisation. Denaturation converts 3' adapted double- stranded nucleic acids into single nucleic acid strands.
  • the population of double-stranded nucleic acids may be denatured by any convenient method following the addition of the 3' adaptor sequence. For example, the nucleic acids may be denatured by heating or treatment with a chemical denaturant .
  • nucleic acids that are sequenceable are isolated from nucleic acids that are non-sequenceable (i.e. molecules not
  • nucleic acids that comprise adaptors at both ends may be isolated, separated or removed from other nucleic acids.
  • immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • the immobilised nucleic acids may be interrogated directly.
  • nucleic acids may be released from the solid support, following washing.
  • nucleic acid strands that comprise the adaptor at the 3' end i.e. sequenceable nucleic acids
  • 3' adapted nucleic acids may then be separated from nucleic acid strands cleaved by the bisulfite treatment and lacking a 3' adaptor.
  • the nucleic acid strands are immobilised before the double stranded nucleic acid is regenerated by primer extension.
  • the population of nucleic acid strands having a 3' adaptor sequence may be immobilised on a solid support following step (iii) .
  • a method of preparing a nucleic acid library may comprise;
  • the nucleic acid strands may be immobilised through a binding tag that is linked to the adaptor sequence for example via a chemical linker.
  • the binding tag may be linked to the adaptor sequence, such that addition of the adaptor sequence, for example by ligation of the adaptor oligonucleotide, links the binding tag to the nucleic acid strands .
  • the population of nucleic acids may be immobilised following primer extension and the generation of the complementary strand.
  • the population of nucleic acids comprising the first adaptor may be immobilised on a solid support following step (v) .
  • the nucleic acids having the first adaptor are immobilised through a binding tag that is linked to the regenerated complementary strand.
  • the binding tag may be covalently linked to the oligonucleotide primer, such that hybridisation of the oligonucleotide primer and subsequent
  • a method of preparing a nucleic acid library may comprise;
  • the capture member and the binding tag may form a specific binding pair.
  • Suitable specific binding pairs may include antibody/immunogenic epitope, such as anti-digoxigenin antibody/digoxigenin; glutathione S-transferase/glutathione; and biotin/biotin binding protein.
  • the binding tag may be an antigen, such as digoxigenin, glutathione, or biotin and the capture member may be an antibody, such as an anti-digoxigenin antibody, glutathione-S-transferase , or a biotin-binding protein, such as streptavidin, avidin, anti-biotin antibody or neutravidin, respectively.
  • the tag is biotin and the capture member is streptavidin.
  • the nucleic acids may then be released from the solid support using any convenient technique to produce a nucleic acid library.
  • Suitable reducing agents include phosphines (e.g.: TCEP) , thiols (e.g.: DTT, EDT) and metal-ligand complexes, including
  • organometallic Ru-, Ir-, Cr-, Rh- and Co- complexes may include organometallic ruthenium (II)
  • ruthenium (II) polypyridine complexes for example ruthenium (II) polypyridine complexes, tris (bipyridine ) ruthenium ( II ) (Ru(bpy) 3 2+ ) and salts thereof, including Ru(bpy) 3 Cl 2 .
  • Other suitable metal-ligand complexes may include organometallic iridium (II) complexes for example iridium polypyridine complexes, such as Ir (ppy) 2 ( dtb-bpy) +, where ppy is phenylpyridine and dtb-bpy is 4, ' -di-tert-butyl-2 , 2 ' -bipyridine, and salts thereof.
  • the linker may comprise a protected hemiaminal ether site.
  • Protected hemiaminal ether sites may be cleaved by removal of the amine protecting group, followed by spontaneous hemiaminal ether cleavage (reaction 2 in Figure 2) .
  • Suitable protecting groups include allyl or allyl carbamates, which may be cleaved using transition metals with water soluble ligands, e.g. Pd with water soluble phosphine ligands); sulfmoc, which may be cleaved with a mild base, e.g.
  • the linker may comprise a phosphine containing site.
  • Phosphine containing sites for example comprising the structure shown in reaction 3 of Figure 2, may be cleaved by the addition of an azide reagent, for example an alkyl or aryl azide, such as benzyl azide.
  • an azide reagent for example an alkyl or aryl azide, such as benzyl azide.
  • the Staudinger aza-ylid generated reacts intramolecularly with an ester to release the captured DNA.
  • containing sites may be cleaved by vicinal elimination of silicon in the presence of fluoride ions, such as KF and tetra-n-butylammonium fluoride (TBAF) (reaction 4 in Figure 2) .
  • fluoride ions such as KF and tetra-n-butylammonium fluoride (TBAF) (reaction 4 in Figure 2) .
  • the linker may comprise a disulfide site.
  • Disulfide sites may be cleavage by reduction with phosphines, such as TCEP or thiols, such as DTT.
  • the linker may comprise a cyanoethyl site. Cyanoethyl sites may be cleaved under basic conditions, such as NH 3 or 10% K 2 C0 3 .
  • Suitable photocleavable sites are well known in the art.
  • an orthonitrobenzyl group may be cleaved by UV at 365 nm.
  • R x and R 2 may be selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or
  • the nucleic acids may be released from the solid support by chemical cleavage of the cleavage site in the linker. This may, for example, release the nucleic acid from a nucleic acid-binding tag-capture member complex. Cleavage of the linker separates the nucleic acid from the binding tag which remains bound to the capture member. immobilised
  • step (ix) above may comprise;
  • nucleic acid library comprising nucleic acid strands having a first adaptor at a first end and a second adaptor at a second end.
  • the immobilised nucleic acids may be denatured, for example using a denaturant such as NaOH, in accordance with standard techniques.
  • nucleic acid manipulation including fragmentation, end-repair, ligation, A-tailing, and primer extension as described herein, are known in the art for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al . , 2001, Cold Spring Harbor Laboratory Press; Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . John Wiley & Sons, 1992) .
  • Methods described herein may comprise interrogating the nucleic acids in the library to identify one or more bases .
  • a method may comprise sequencing one or more,
  • nucleic acids may be sequenced using any convenient low or high throughput sequencing technique or platform, including Sanger sequencing, Solexa-Illumina sequencing, Ligation-based sequencing (SOLiDTM), pyrosequencing; Pacific Biosciences single molecule rea time sequencing (SMRTTM) ; and semiconductor array sequencing (Ion TorrentTM) .
  • Suitable protocols, reagents and apparatus for nucleic acid sequencing are well-known in the art and are available
  • the nucleic acids in the library are sequenced without amplification .
  • the nucleic acids in the nucleic acid library may be interrogated by PCR, hybridisation, for example to an array of immobilised probes or other analysis methods.
  • kits for use in the preparation of a nucleic acid library as described herein may comprise;
  • the complementary oligonucleotide and the oligonucleotide primer are hybridisable to the adaptor oligonucleotide and are preferably complementary to all or part of the adaptor oligonucleotide.
  • One of the adaptor oligonucleotide and the oligonucleotide primer may be linked to a binding tag via a cleavable linker. Cleavable linkers and binding tags are discussed above.
  • the kit may further comprise nucleic acid isolation reagents.
  • a kit may comprise 1- ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) or O-ethylhydroxylamine for chemical modification-assisted bisulfite sequencing and 5fC chemical modification-assisted bisulfite
  • EDC carbodiimide hydrochloride
  • O-ethylhydroxylamine for chemical modification-assisted bisulfite sequencing and 5fC chemical modification-assisted bisulfite
  • oligonucleotides may be made synthetically by standard methods .
  • the kit may include instructions for use in a method of preparation of a nucleic acid library as described above.
  • Figure 14 The effect of GC content on depth of coverage
  • (a) A genome browser view showing the coverage obtained across the P. berghei apicoplast for both methods. While near constant for REBUiLT, there are distinct read pile-ups in PCR-BS that appear to track GC content
  • oligonucleotide primer is linked to a binding tag and the method comprises binding the tag to a capture member immobilised on a solid support thereby immobilising the population of nucleic acids.
  • nucleic acid library comprising nucleic acid strands having a first adaptor sequence at a first end and a second adaptor sequence at a second end.
  • genomic DNA molecules are genomic DNA molecules .
  • nucleic acids from one of: a cell, a sample of cells, and a biological fluid sample,
  • a method according to embodiment 1.31 comprising modifying the 3' ends of the nucleic acids.
  • 1.33 A method according to embodiment 1.32 wherein the 3' ends are modified by the addition of an overhanging adenine residue.
  • 1.34 A method according to any one of the preceding embodiments wherein the double-stranded nucleic acids in the population comprise a one base 3' overhang consisting of an adenine residue.
  • 1.38 A method according to embodiment 1.36 or embodiment 1.37 comprising ligating the complex to the population such that the adaptor oligonucleotide of the complex is covalently linked to the 3' ends of the double-stranded nucleic acids and the complementary oligonucleotide of the complex is not linked to the 5' ends of the double-stranded nucleic acids.
  • 1.39 A method according to embodiment 1.38 wherein the double- stranded nucleic acids in the population lack 5' phosphate groups.
  • said hairpin adaptor comprising a hairpin sequence that links the adaptor sequence and the complementary sequence.
  • hairpin adaptor comprises a first cleavage site at the 3' end of the complementary sequence and a second cleavage site at the 5' end of the adaptor sequence .
  • nucleic acids are treated with bisulfite after ligation of the double-stranded adaptor and before cleavage of the 5' ends.
  • 1.47 A method according to any one of the preceding embodiments comprising modifying the second ends of the double stranded nucleic acids generated by extension of the oligonucleotide primer. 1.48 A method according to embodiment 1.47 comprising adding a 5' phosphate group and a 3' adenine residue to the second end of the double-stranded nucleic acids .
  • a method according to embodiment 1.48 comprising;
  • a hairpin adaptor comprising an adaptor sequence and a a complementary sequence
  • kit according to any one of embodiments 1.50 to 1.52 further comprising a bisulfite reagent
  • kit according to any one of embodiments 1.50 to 1.53 further comprising one or more nucleic acid isolation reagents.
  • kits according to any one of embodiments 1.50 to 1.54 further comprising one or more end-repair reagents.
  • 1.56 A kit according to embodiment 1.55 wherein the end-repair reagents do not include a 5' kinase.
  • kits according to any one of embodiments 1.50 to 1.56 further comprising one or more end-modification reagents.
  • kits according to any one of embodiments 1.50 to 1.57 further comprising one or more cleavage reagents for cleavage of the hairpin primer .
  • kits according to embodiment 1.58 wherein the cleavage reagents comprise formamidopyrimidine [fapy]-DNA glycosylase.
  • a kit according to any one of embodiments 1.50 to 1.59 further comprising a solid support.
  • kits according to any one of embodiments 1.50 to 1.60 further comprising one or more sequencing reagents.
  • the adaptor sequence is ligated to the 3' ends of the double-stranded nucleic acids and the complementary sequence is ligated to the 5' ends of the double-stranded nucleic acids, and cleaving the nucleic acids to remove the complementary sequence .
  • a method according to any one of embodiments 1.63 to 1.70 comprising isolating the double-stranded nucleic acids having an adaptor at a first end and a second adaptor at a second end.
  • the recovery after bisulfite treatment (ReBuilT) method begins with fragmentation, end repair and A-tailing.
  • ddT dideoxythymidine
  • the presence of a 3' ddT prevents ligation to the 5' end of the insert DNA, resulting in a single stranded directional ligation to the 3' insert terminus
  • a primer extension step with a high fidelity uracil tolerant polymerase is performed to generate blunt ended double stranded DNA, which is immobilized on streptavidin coated magnetic beads via the biotin label.
  • the immobilized DNA is end repaired and A-tailed before ligation of a fully complementary adapter.
  • To generate sequenceable fragments we copy the bisulfite- converted strands by single primer extension. These new strands contain only the canonical DNA bases (A, T, G and C) , which is necessary as standard next-generation sequencing platforms are incompatible with uracil containing DNA.
  • A, T, G and C canonical DNA bases
  • the first directional ligation prevents the formation of adapter dimers, a common sequencing contaminant that lead to non-insert sequencing reads .
  • Adapter dimers forming during the second ligation have no impact on library composition, as they are completely removed during washing of the beads.
  • the immobilization on beads enables near lossless library manipulation.
  • the ReBuilT libraries retain approximately double the percentage of raw data for methylation calling when compared to the PCR-BS libraries.
  • the ReBuilT method therefore, yields considerably more useable data, which reduces the sequencing power required for methylation analysis.
  • duplication rate ( Figure 16) .
  • the ReBuilT libraries were found to have an average duplication rate of 16%.
  • PCR-BS sample has almost double the duplication rate of 30%, which is a cumulative effect of amplification duplicates and extremely uneven coverage. Uneven coverage leads to peaks and troughs in read depth, which will locally raise or lower the expected duplication rate.
  • PCR-BS samples exhibit a strong preference for the relatively GC rich windows .
  • the preference for a balanced base composition has the potential to introduce two types of artifacts when analyzing the methylome. Firstly, the quantification of methylation levels will be affected. As 5mC bases are not converted to thymine during bisulfite treatment, DNA fragments containing methylated loci will tend to have a higher GC content. As GC content can clearly affect amplification efficiency, it is no longer correct to determine the methylation level at a site with the (C/C+T) formula. Secondly, certain biological features display
  • Apicomplexan parasites of which Plasmodium is one, have a non- photosynthetic relict plastid called the apicoplast that codes for proteins that participate in lipid biosynthesis and iron metabolism.
  • This organelle contains multiple copies of a 35 kb genome, and it has been suggested is unmethylated (Ponts, N. et al . Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum. Cell Host Microbe 14, 696-706 (2013) ) . We determined the average number of genome copies to be 5.5, and detected significant methylation along its sequence.
  • 500ng of human genomic DNA was prepared and BS treated using the PCR free method described above. 35 bp paired ends were sequenced on an Illumina Miseq instrument.
  • oligomer ODNlb was purified by ethanol precipitation, and resuspended in 10 mM Tris, 50 mM NaCl.
  • the underlined six-nucleotide portion of oligomer ODNlb was varied to give different adapter barcodes. All cytosines in ODNlb were replaced with 5mC to retain the adapter sequence following bisulfite conversion.
  • Adapter pairs were annealed in a thermocycler (95 °C for 10 minutes, cooling to 70 °C over 10 minutes, holding at 70 °C for 10 minutes and then slowly cooling to RT at 0.1 °C s _1 ) to give 25 ⁇ solutions in 10 mM Tris-HCl pH 7. , 50 mM NaCl.
  • Annealing ODNla and ODNlb generated adapter pair 1; annealing ODN2a + ODN2b generated adapter pair 2.

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Abstract

La présente invention concerne la préparation d'acides nucléiques, par exemple, d'acides nucléiques traités au bisulfite, pour l'analyse de marques portées par des cytosines modifiées. L'invention concerne également un procédé de préparation d'une bibliothèque d'acides nucléiques traités au bisulfite comprenant une procédure de ligature en deux étapes, où un premier adaptateur est ajouté avant un traitement au bisulfite et un second adaptateur est ajouté par la suite.
PCT/GB2015/050871 2014-03-24 2015-03-24 Procédé de préparation d'acides nucléiques WO2015145133A1 (fr)

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Cited By (11)

* 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
WO2020106893A1 (fr) * 2018-11-21 2020-05-28 Karius, Inc. Procédés, systèmes et compositions de bibliothèque directe
US20200248248A1 (en) * 2016-04-07 2020-08-06 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnostics by sequencing 5-hydroxymethylated cell-free dna
WO2021097252A1 (fr) * 2019-11-13 2021-05-20 Bradley Bernstein Dosage de méthylation et leurs utilisations
CN113227468A (zh) * 2018-11-21 2021-08-06 卡里乌斯公司 感染性疾病的检测和预测
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US11692224B2 (en) 2016-03-25 2023-07-04 Karius, Inc. Synthetic nucleic acid spike-ins
US12024750B2 (en) 2018-04-02 2024-07-02 Grail, Llc Methylation markers and targeted methylation probe panel

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009132315A1 (fr) * 2008-04-24 2009-10-29 Life Technologies Corporation Procédé de séquençage et d'élaboration de la carte d'acides nucléiques cibles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009132315A1 (fr) * 2008-04-24 2009-10-29 Life Technologies Corporation Procédé de séquençage et d'élaboration de la carte d'acides nucléiques cibles

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
AIRD DANIEL ET AL: "Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries", GENOME BIOLOGY, BIOMED CENTRAL LTD., LONDON, GB, vol. 12, no. 2, 21 February 2011 (2011-02-21), pages R18, XP021091793, ISSN: 1465-6906, DOI: 10.1186/GB-2011-12-2-R18 *
BOOTH MICHAEL J ET AL: "Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine", NATURE PROTOCOLS, NATURE PUBLISHING GROUP, GB, vol. 8, no. 10, 1 October 2013 (2013-10-01), pages 1841 - 1851, XP009175350, ISSN: 1750-2799, [retrieved on 20130905] *
HOEIJMAKERS WIETEKE A M ET AL: "Linear amplification for deep sequencing", NATURE PROTOCOLS, NATURE PUBLISHING GROUP, GB, vol. 6, no. 7, 1 July 2011 (2011-07-01), pages 1026 - 1036, XP009177348, ISSN: 1750-2799, [retrieved on 20110623] *
M. J. BOOTH ET AL: "Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution", SCIENCE, vol. 336, no. 6083, 18 May 2012 (2012-05-18), pages 934 - 937, XP055064913, ISSN: 0036-8075, DOI: 10.1126/science.1220671 *
R. LISTER ET AL: "Finding the fifth base: Genome-wide sequencing of cytosine methylation", GENOME RESEARCH, vol. 19, no. 6, 9 March 2009 (2009-03-09), pages 959 - 966, XP055190057, ISSN: 1088-9051, DOI: 10.1101/gr.083451.108 *
RYAN LISTER ET AL: "Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis", CELL, vol. 133, no. 3, 1 May 2008 (2008-05-01), pages 523 - 536, XP055190066, ISSN: 0092-8674, DOI: 10.1016/j.cell.2008.03.029 *
RYAN LISTER ET AL: "Supplemental data: Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis", CELL, vol. 133, no. 3, 1 May 2008 (2008-05-01), pages 523 - 536, XP055190502, ISSN: 0092-8674, DOI: 10.1016/j.cell.2008.03.029 *
SHAWN J. COKUS ET AL: "Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning", NATURE, vol. 452, no. 7184, 17 February 2008 (2008-02-17), pages 215 - 219, XP055190064, ISSN: 0028-0836, DOI: 10.1038/nature06745 *
SHAWN J. COKUS ET AL: "Supplementary Information: Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning", NATURE, vol. 452, no. 7184, 17 February 2008 (2008-02-17), pages 215 - 219, XP055190075, ISSN: 0028-0836, DOI: 10.1038/nature06745 *
TANAKA K ET AL: "Degradation of DNA by bisulfite treatment", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, AMSTERDAM, NL, vol. 17, no. 7, 1 April 2007 (2007-04-01), pages 1912 - 1915, XP026265824, ISSN: 0960-894X, [retrieved on 20070312], DOI: 10.1016/J.BMCL.2007.01.040 *

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US20200248248A1 (en) * 2016-04-07 2020-08-06 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnostics by sequencing 5-hydroxymethylated cell-free dna
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US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
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