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WO2010064040A1 - Procédé pour séquençage de polynucléotides - Google Patents

Procédé pour séquençage de polynucléotides Download PDF

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Publication number
WO2010064040A1
WO2010064040A1 PCT/GB2009/051635 GB2009051635W WO2010064040A1 WO 2010064040 A1 WO2010064040 A1 WO 2010064040A1 GB 2009051635 W GB2009051635 W GB 2009051635W WO 2010064040 A1 WO2010064040 A1 WO 2010064040A1
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Prior art keywords
sequencing
library
genome
pcr
adapters
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PCT/GB2009/051635
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English (en)
Inventor
Iwanka Kozarewa
Daniel John Turner
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Genome Research Ltd
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Genome Research 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to methods for DNA amplification and sequencing.
  • a single lane of an lllumina Genome Analyzer (GA) flowcell 7 can currently yield > 400 x 10E6 bases of purity filtered (PF) sequence data in a 7 day paired end run. This would represent >18 x coverage of the genome of the 23Mb reference P. falciparum clone, 3D7 [6].
  • PF purity filtered
  • the lllumina library preparation pipeline is a multi-step process: adapters are ligated onto fragmented, end-repaired, A-tailed sample DNA, via a 3' T-overhang.
  • the structure of the adapters ensures that whenever they ligate to both ends of a template strand, each strand receives a unique adapter sequence at either end.
  • the lllumina library preparation pipeline exploits the polymerase chain reaction (PCR) [7].
  • PCR polymerase chain reaction
  • the lllumina library prep uses carefully designed universal PCR primers, which allow for more optimal, simultaneous amplification of all loci, and facilitate amplification of complex template pools [7].
  • template concentrations in the PCR There is a narrow range of template concentrations in the PCR that will give clean libraries with adequate representation: too high a template concentration often generates an unexpected peak with an apparently higher molecular weight peak; too low a mass of template DNA in the PCR causes an increased incidence of PCR duplicates in the resulting sequences.
  • the PCR step is still sensitive to biases, particularly when the template to be amplified has particularly high AT content.
  • the present invention addresses the issue of optimising sequencing techniques.
  • the present invention relates to a method for sequencing of polynucleic acids, the method comprising the steps of
  • Figure 1 shows Distribution of genome sequence coverage
  • a) The distribution of sequence coverage across the unmasked genome is shown with various datasets with or without the PCR step
  • Figure 2. shows 'No-PCR library preparation'.
  • Figure 3 shows GC content and depth of coverage.
  • Figure 4 shows frequencies of duplicate sequences.
  • the malaria sequencing programme at the Wellcome Trust Sanger Institute aims to sequence hundreds of cell lines, including clinical isolates collected from wild environment.
  • Plasmodium falciparum 3D7 the same strain as the reference genome 6, on the lllumina Genome Analyzer, with the intention of correcting base errors in the reference. This was followed by several more sequencing runs for a variety of malaria strains. Sequencing using the standard lllumina pipeline was successful in throughput, but quality of read mapping against the reference was very poor. The short reads were mapped to the reference using the modified SSAHA program [13] (see methods).
  • the lllumina library preparation generally introduces tails onto library DNA in a 2-step process. Firstly, adapters, essentially consisting of the sequencing primer annealing sequences, are ligated on. Then an additional section is added, by PCR, which facilitates hybridisation of library fragments to the flowcell. But, even though the number of cycles of PCR amplification is kept low (10 cycles) 7, it is a source of duplicate sequences, and amplification bias, and struggles with base compositions that lie at the extremes of low or high GC content [14].
  • a library of polynucleic acid (such as DNA) fragments are generated, for example, by random shearing and joined to a pair of oligonucleotides in a forked adaptor configuration.
  • the ligated products are amplified using two oligonucleotide primers, resulting in double-stranded blunt-ended material with a different adaptor sequence on either end.
  • Polynucleic acid fragments prepared as in 'A' are denatured and single strands are annealed to complementary oligonucleotides on the flowcell surface.
  • a new strand is copied from the original strand in an extension reaction that is primed from the 3' end of the surfacebound oligonucleotide; the original strand is then removed by denaturation.
  • the adapter sequence at the 3' end of each copied strand is annealed to a new surface-bound complementary oligonucleotide, forming a bridge and generating a new site for synthesis of a second strand.
  • the polynucleic acid in each cluster is linearized by cleavage within one adapter sequence and denatured, generating single-stranded template for sequencing by synthesis to obtain a sequence read (read 1 ).
  • read 1 a sequence read
  • the products of read 1 are removed by denaturation, the template is used to generate a bridge, the second strand is re-synthesized and the opposite strand is then cleaved to provide the template for the second read (read 2).
  • the present invention omits the step of PCR amplification and is thus able to improve on known methods.
  • the present invention relates to a method for sequencing of polynucleic acids, the method comprising the steps of:
  • the invention relates to a method for preparation of a DNA library, the method comprising the step of ligating a library of polynucleic acid fragments to adapters which facilitate hybridisation of the library fragment to a solid support to provide a surface bound polynucleic acid, wherein the adapters comprise both a region suitable for sequencing primer annealing and a region suitable for attachment to a solid support.
  • the present invention uses a solid support amplification step (cluster amplification) to select for fully ligated template strands, rather than a PCR step.
  • cluster amplification solid support amplification step
  • the method comprises ligating a library of polynucleic acid fragments from a genome that has an uneven nucleotide composition, for example having a mean AT content of > 60%, >70%, > 80%, or higher, or a genome having a mean GC content of > 60%, >70%, > 80%, or higher.
  • polynucleic acid is DNA.
  • the solid support is glass or a plastics material, and may be a flat surface or curved surface, such as a bead.
  • the adapter comprises a region suitable for sequencing primer annealing and a region suitable for attachment to a solid support. In one aspect these regions are separate, such that the target for sequencing may be accessible for productive sequencing even when the adapter is bound to a solid support.
  • one of the adapters is equivalent in design (and may be identical) to one of the 2 "lllumina" PCR primers used to amplify the ligated products of the prior art method outlined above, whereas the other adapter is the reverse complement of the other lllumina PCR primer.
  • Prior art methods are for example as disclosed in the lllumina methodology used in reference [7] and as generally disclosed in see http://www.illumina.com.
  • the library polynucleic acids fragments are ligated on appropriate adapters after end repair and A-tailing, suitably using standard lllumina protocols (see http://www.illumina.com).
  • the adapters are partially non-complementary, thus ensuring that a different adapter sequence is added to either end of the template strands.
  • the adapters consist of a pair of oligonucleotides that have a region of nucleotides that are complementary, the remainder being non-complementary.
  • the oligonucleotides may be 40 or more nucleotides in length, such as 50, 60, 70 or 80 nucleotides or more.
  • Oligonucleotides may have a complementary region of 10 or more nucleotides, such as 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides.
  • the pair of oligonucleotides are mixed together in substantially eqimolar or equimolar quantity, in a suitable buffer, and suitably are phosphorylated at their 5' ends using, for example, an enzyme such as T4 polynucleotide kinase.
  • the oligonucleotides are then hybridized to one another.
  • the phosphrylation method is allowed to proceed for a suitable length of time, then the temperature is raised so as to denature the kinase enzyme, and to disrupt any secondary structure within the oligonucleotides.
  • the temperature is then lowered to room temperature slowly, so that the complementary sections of the two oligonucleotides can anneal together, forming a ⁇ -shaped' structure.
  • the complementary section of one strand of the adapter has an additional T nucleotide at its 3' end, so that after annealing, a T-overhang is formed, which allows more efficient ligation to a suitably prepared target DNA.
  • This T-nucleotide is attached to the rest of that oligonucleotide strand via a phosphorothioate linkage, which confers resistance against exonuclease digestion. This prevents removal of this T-nucleotide, and thus prevents blunt ended self-ligation of adapter molecules.
  • One strand of the adapter has a nucleotide region at its 5' end, which facilitates hybridisation to oligonucleotides on a solid surface.
  • the remaining nucleotides of this strand consist of a region to which a sequencing primer can hybridise or which otherwise facilitates sequencing primer hybridisation.
  • the other strand has a nucleotide region at its 3' end, which facilitates hybridisation to oligonucleotides on a solid surface.
  • the remaining nucleotides of this strand consist of a region to which a sequencing primer can hybridise.
  • the adapter molecule can be ligated to any double stranded DNA template that has been prepared in such a way as to have a single protruding A-nucleotide at the 3' termini of both strands.
  • adapter - template complexes are suitably run in an agarose gel, for the purpose of size selection. This allows a gel slice to be taken, representing a mixture of ligated template molecules, all within a particular size range. It also allows removal of adapter dimers - i.e. those that have ligated to one another. DNA is extracted from the gel and is quantified by qPCR and then sequenced.
  • the invention also relates to mixtures of oligonucleotides as described above and to kits comprising such pairs of oligonucleotides, separately or in the form of ligated adapter molecules. .
  • a portion of the library is quantified using qPCR after ligation of the adaptors.
  • the qPCR is used to quantify only those strands that have an adapter at either end.
  • the amplification of the surface bound polynucleic acid fragment by multiple cycles of annealing, extension and denaturation is bridge amplification, wherein during the annealing step of the amplification cycle the extension product from one bound primer forms a bridge to the other bound primer.
  • the amplification step uses well known conditions for annealing, extension and denaturation.
  • sequence information obtained in step 3 is used for genomic DNA analysis.
  • step 3 is carried out using the process of reversible terminator chemistry as disclosed in reference [7].
  • the method is as described in reference [7] herein, with the exception of the PCR amplification step prior to attachment of the polynucleic acids to the solid support.
  • the method thus comprises the following steps:
  • DNA fragments are joined to a pair of adapter oligonucleotides, suitably in a forked adaptor configuration wherein one end of the adaptor strand is not complementary to the other end of the other adaptor strand.
  • DNA fragments prepared as in '1' are denatured and single strands are annealed to complementary oligonucleotides on a solid support, suitably a flowcell surface.
  • a new strand is copied from the original strand in an extension reaction that is primed from the 3' end of the surfacebound oligonucleotide; the original strand is then removed by denaturation.
  • the adapter sequence at the 3' end of each copied strand is annealed to a new surface-bound complementary oligonucleotide, forming a bridge and generating a new site for synthesis of a second strand.
  • each cluster is linearized by cleavage within one adapter sequence and denatured, generating single-stranded template for sequencing by synthesis to obtain a sequence read (read 1 ).
  • read 1 sequence read
  • paired-read sequencing optional step
  • the products of read 1 are removed by denaturation
  • the template is used to generate a bridge
  • the second strand is re- synthesized
  • the opposite strand is then cleaved to provide the template for the second read (read 2)
  • A_adapter_t (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC CGATC*T, * indicates phosphorothioate)
  • A_adapter_b (GATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTC TTCTGCTTG).
  • A_adapter_t contained a phosphorothioate modification to resist exonuclease digestion at the T-overhang.
  • 40 ⁇ M oligos were phosphorylated at the 5' end by 1 unit / ⁇ l T4 polynucleotide kinase in 1x T4 ligase buffer (both New England
  • Genomic DNA was quantified by NanoDrop. We fragmented 4.5 ⁇ g DNA to approximately 200bp using Covaris Adaptive Focused Acoustics technology, using the settings: 5% Duty Cycle; Intensity 10; 200 Cycles per burst over the course of 12 minutes (using Covaris product number 520031 , 300 ⁇ l, 6 x 32mm Round bottom glass tube, with no fibre, with crimp-cap system. It is also possible to use Covaris product number 520052, 100 ⁇ l, 6 x 16mm Round bottom glass tube and AFA fibre with crimp-cap system over a course of 90 seconds.
  • Standard sequencing libraries were prepared following the manufacturer's recommended protocol, except that genomic DNA was fragmented using Covaris Adaptive Focused Acoustics rather than nebulisation, as described above.
  • genomic DNA was fragmented using Covaris Adaptive Focused Acoustics rather than nebulisation, as described above.
  • SSAHA Sequence Search and Alignment by Hashing Algorithm
  • the alignment files were further processed for SNP detection using a variation detection pipeline ssaha_pileup ( ftp://ftp.sanger.ac.uk/pub/zn1/ssaha_pileup/). It is expected that the high sensitivity offered by the alignment tool should improve read mapping, particularly, for those extremely AT biased genomes such as Plasmodium falciparum.
  • Pair-ending Solexa typed data provides challenging, but exciting prospects for de novo assemblies, where requirement for read coverage across the genome is more than that for variation detections.
  • every base has to be covered several times from raw reads in order to make a consensus.
  • the assembly process consists of two distinctive steps: read extension and whole genome assembly. Firstly, raw sequencing Solexa reads are extended into segments of consensus sequences each with a maximum length of 2 kb. Sequence extension starts from kmer seeds which are randomly sampled to ensure overlaps among extended segments. To obtain genome assemblies, the new data set of the extended sequences with 10-15X coverage is processed and assembled using the Phusion assembler [16] - the previously-developed capillary read assembly pipeline.
  • Figure 1(a) shows the distribution of fraction of unmasked genome against coverage depth for 7 different datasets. The peak of fraction distribution from the no-PCR data is in close agreement with the read depth, a feature which does not occur for other four sets of data using the standard lllumina library prep pipeline.
  • the extreme AT bias of the Plasmodium falciparum genome may be correlated to the sequence GC content at zero coverage, shown in Figure 3 for the two no-PCR datasets. If this is the case, those sequences with zero coverage are likely to be repetitive segments, where short reads cannot be uniquely assigned even using the read pairs.
  • read coverage is low (e.g. less than 10)
  • the curves observed in Figure 3 are notably different using various library preparations.
  • the value of GC content decreases with an increase in read coverage for the no-PCR datasets, indicating that it is more difficult to place reads on lower GC sequences.
  • PCR duplicates are a major concern in lllumina sequencing and, from our previous experience, are generally dependent upon the quality of library preparations. Presumably, duplicates are more abundant in smaller genome libraries because they represent a higher percentage of the total number of possible fragments. Reducing duplicates would be beneficial for all sizes of genomes, both in lowering costs and allowing improved read mapping.
  • a duplicate is regarded as a number of reads which share exactly the start and end matching locations and the read number is defined as duplication depth or number of exactly duplicated matches.
  • the duplication rate is very low for the two no-PCR datasets and high for STD-368 and STD-245 as the tails on the curves extend far. It is interesting to note that the duplication rate looks high for STD-883 from a PF clinical run, but in fact it is normal judged from the distribution of read duplication.
  • the no-PCR duplication frequency is caused by noise in the cluster detection and analysis software, and by the fact that each double stranded template is capable of forming two identical clusters.
  • FIG. 1 Distribution of genome sequence coverage, a) The distribution of sequence coverage across the unmasked genome is shown with various datasets with or without the PCR step, b) Accumulated portion of unmasked genome at different depth of coverage
  • FIG. 1 No-PCR library preparation, a) Partially complementary ('Y-shaped') adapters with a 3' T overhang are ligated onto fragmented, end-repaired, 3' A-tailed DNA. This stricture ensures that each strand of the template molecule receives a different adapter sequence at each end.
  • the adapter strands each consist of two sections: FP1 or FP2', which allow hybridisation of the ligated template molecules to the flowcell surface, and R1 or R2', which allow hybridisation of sequencing primers, b)
  • the standard library prep uses PCR to enrich for fully ligated templates. Only these templates amplify on the flowcell surface. With the no-PCR approach, the flowcell itself is used to select for fully ligated template molecules.
  • Unligated template molecules do not anneal to the flowcell oligos, whereas semi-ligated template strands hybridise and will be copied in the initial extension reaction, but without the adapter at the other end, these are unable to participate in bridge amplification. If, by virtue of coincidental sequence similarity, a semi-ligated template does amplify to form a cluster, it will not sequence from the unligated end because sequencing primer hybridization will not be possible.
  • FIG. 1 GC content and depth of coverage. GC content of genome sequence is plotted against the depth of genome coverage with various datasets with or without the PCR step.
  • the lllumina library preparation pipeline introduces tails onto library DNA in a 2-step process. Firstly, adapters, essentially consisting of the sequencing primer annealing sequences, are ligated on. Then an additional section is added, by PCR, which facilitates hybridisation of library fragments to the flowcell. But, even though the number of cycles of PCR amplification is kept low (10 cycles) [7], it is a source of duplicate sequences, and amplification bias, and struggles with base compositions that lie at the extremes of low or high GC content [14]. The consequences are that each run becomes less efficient and that assembly, mapping and SNP detection are made more complicated than necessary.
  • the structure of the adapters ensures that all fully ligated templates receive a different sequence at each end, though because the efficiency of ligation is not 100%, many template strands will receive no adapters, or will only be partially ligated.
  • templates cannot amplify on the flowcell surface, and in this way, the cluster amplification step performs the enrichment that is otherwise provided in the PCR.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps
  • combination refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne un procédé de séquençage d'acides polynucléiques, ce procédé comprenant les opérations consistant: (i) à ligaturer une échantillothèque de fragments d'acide polynucléique sur des adaptateurs qui facilitent l'hybridation du fragment d'échantillothèque sur un support solide de façon à donner un acide polynucléique lié à une surface; (ii) à prendre le fragment d'acide polynucléique lié à une surface et à l'amplifier en plusieurs cycles de renaturation, extension et dénaturation (amplification de grappe); et (iii) à séquencer les acides polynucléiques amplifiés, les fragments d'acides polynucléiques ligaturés sur les adaptateurs n'étant pas amplifiés préalablement à la liaison au support solide.
PCT/GB2009/051635 2008-12-02 2009-12-02 Procédé pour séquençage de polynucléotides Ceased WO2010064040A1 (fr)

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GBGB0821981.8A GB0821981D0 (en) 2008-12-02 2008-12-02 Method for use in polynucleotide sequencing
GB0821981.8 2008-12-02

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Cited By (3)

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EP2855707A4 (fr) * 2012-05-31 2016-01-06 Univ Texas Procédé de séquençage précis d'adn
CN106795650A (zh) * 2014-09-26 2017-05-31 深圳华大基因股份有限公司 Pf快速建库方法及其应用
CN111383715A (zh) * 2013-04-17 2020-07-07 先锋国际良种公司 用于在基因组中表征dna序列组成的方法

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2855707A4 (fr) * 2012-05-31 2016-01-06 Univ Texas Procédé de séquençage précis d'adn
AU2013267609B2 (en) * 2012-05-31 2018-06-28 Board Of Regents, The University Of Texas System Method for accurate sequencing of DNA
AU2013267609C1 (en) * 2012-05-31 2019-01-03 Board Of Regents, The University Of Texas System Method for accurate sequencing of DNA
US10870882B2 (en) 2012-05-31 2020-12-22 Board Of Regents, The University Of Texas System Method for accurate sequencing of DNA
US11584964B2 (en) 2012-05-31 2023-02-21 Board Of Regents, The University Of Texas System Method for accurate sequencing of DNA
US12139757B2 (en) 2012-05-31 2024-11-12 Board Of Regents, The University Of Texas System Method for accurate sequencing of DNA
CN111383715A (zh) * 2013-04-17 2020-07-07 先锋国际良种公司 用于在基因组中表征dna序列组成的方法
CN106795650A (zh) * 2014-09-26 2017-05-31 深圳华大基因股份有限公司 Pf快速建库方法及其应用
CN106795650B (zh) * 2014-09-26 2021-03-09 深圳华大基因股份有限公司 Pf快速建库方法及其应用

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