WO2007073165A1 - Method for high-throughput aflp-based polymorphism detection - Google Patents

Method for high-throughput aflp-based polymorphism detection Download PDF

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Publication number
WO2007073165A1
WO2007073165A1 PCT/NL2006/000648 NL2006000648W WO2007073165A1 WO 2007073165 A1 WO2007073165 A1 WO 2007073165A1 NL 2006000648 W NL2006000648 W NL 2006000648W WO 2007073165 A1 WO2007073165 A1 WO 2007073165A1
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Prior art keywords
adaptor
aflp
dna
sequencing
restriction
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PCT/NL2006/000648
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French (fr)
Inventor
Michael Josephus Theresia Van Eijk
Anker Preben SØRENSEN
Marco Gerardus Maria Van Schriek
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Keygene N.V.
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Priority to JP2008547127A priority Critical patent/JP5452021B2/en
Priority to ES06835670T priority patent/ES2391837T3/en
Priority to EP06835670A priority patent/EP1966393B1/en
Priority to DK06835670.8T priority patent/DK1966393T3/en
Priority to CN200680051561.8A priority patent/CN101374963B/en
Priority to US12/158,040 priority patent/US8481257B2/en
Priority to EP18174221.4A priority patent/EP3404114B1/en
Application filed by Keygene N.V. filed Critical Keygene N.V.
Publication of WO2007073165A1 publication Critical patent/WO2007073165A1/en
Priority to US13/666,385 priority patent/US8815512B2/en
Priority to US14/274,591 priority patent/US9334536B2/en
Priority to US14/318,352 priority patent/US8911945B2/en
Priority to US14/550,805 priority patent/US9062348B1/en
Priority to US14/699,891 priority patent/US9328383B2/en
Priority to US15/136,224 priority patent/US9777324B2/en
Priority to US15/366,417 priority patent/US9702004B2/en
Priority to US15/683,252 priority patent/US10106850B2/en
Priority to US16/165,645 priority patent/US20190144938A1/en
Priority to US16/517,502 priority patent/US11008615B2/en

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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to the fields of molecular biology and genetics.
  • the invention relates to rapid discovery, detection and large-scale genotyping of polymorphisms in a nucleic acid sample or between samples.
  • the identified polymorphisms may be used as genetic markers.
  • Genomic DNA holds the key to identification, diagnosis and treatment of diseases such as cancer and Alzheimer's disease.
  • exploration of genomic DNA may provide significant advantages in plant and animal breeding efforts, which may provide answers to food and nutrition problems in the world.
  • Markers i.e. genetic markers
  • a genetic typing method i.e. to connect a phenotypic trait to the presence, absence or amount of a particular part of DNA (gene) .
  • AFLP AFLP
  • AFLP technology Zabeau & Vos, 1993; Vos et al., 1995
  • AFLP AFLP technology
  • a cornerstone of AFLP ensures that the number of amplified fragments can be brought in line with the resolution of the detection system, irrespective of genome size or origin.
  • AFLP fragments are commonly carried out by electrophoresis on slab-gels (Vos et al. r 1995) or capillary electrophoresis (van der Meulen et al., 2002) .
  • the majority of AFLP markers scored in this way represent (single nucleotide) polymorphisms occurring either in the restriction enzyme recognition sites used for AFLP template preparation or their flanking nucleotides covered by selective AFLP primers.
  • the remainder of the AFLP markers are insertion/deletion polymorphisms occurring in the internal sequences of the restriction fragments and a very small fraction on single nucleotide substitutions occurring in small restriction fragments ( ⁇ approximately 100 bp) , which for these fragments cause reproducible mobility variations between both alleles; these AFLP markers can be scored co-dominantly without having to rely on band intensities.
  • the AFLP markers therefore constitute the minority of amplified fragments (less than 50 percent but often less than 20 percent) , while the remainder are commonly referred to as constant AFLP fragments.
  • the latter are nevertheless useful in the gel scoring procedure as they serve as anchor points to calculate fragments mobilities of AFLP markers and aid in quantifying the markers for co-dominant scoring.
  • Co-dominant scoring scoring for homo- or heterozygosity
  • AFLP markers currently is restricted to the context of fingerprinting a segregating population. In a panel of unrelated lines, only dominant scoring is possible.
  • Electrophoresis allows unique identification of the majority of amplified fragments based on the combination of restriction enzyme combinations (EC) , primer combinations (PC) and mobility, but ideally, the detection system should be capable of determining the entire sequence of the amplified fragments to capture all polymorphisms . Detection by sequencing instead of mobility determination will increase throughput because:
  • This limitation does not apply to detection by electrophoresis because position information on the gel is available. Accordingly, it is one of the further goals of the present invention provide a method that solves the problem of sample variation or at least reduces the errors caused by sample variation to an acceptable minimum.
  • the present inventors have found that sequencing is within reach for the detection of AFLP and SNP markers with the use of AFLP in certain adapted procedures for high throughput sequencing.
  • the invention thus provides a method or strategy which combines the power and generic applicability of AFLP with certain high throughput sequencing technologies to establish a generically applicable polymorphism scoring system.
  • this strategy the issue of sampling variation is also addressed to ensure genotyping with high accuracy and maximizing chances for datasets with minimal numbers of missing genotypes .
  • polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population.
  • a polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion.
  • a polymorphism includes e.g.
  • SSR simple sequence repeat
  • SNP single nucleotide polymorphism
  • a variation must generally occur in at least 1% of the population to be considered a SNP.
  • SNPs make up e.g. 90% of all human genetic variations, and occur every 100 to 300 bases along the human genome. Two of every three SNPs substitute Cytosine (C) with Thymine (T) . Variations in the DNA sequences of e.g. humans or plants can affect how they handle diseases, bacteria, viruses, chemicals, drugs, etc.
  • Nucleic acid may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes) .
  • the present invention contemplates any deoxyribonucleotide,. ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or, glycosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
  • the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
  • Complexity reduction is used to denote a method wherein the complexity of a nucleic acid sample, such as genomic DNA, is reduced by the generation of a subset of the sample.
  • This subset can be representative for the whole (i.e. complex) sample and is preferably a reproducible subset. Reproducible means in this context that when the same sample is reduced in complexity using the same method, the same, or at least comparable, subset is obtained.
  • the method used for complexity reduction may be any method for complexity reduction known in the art.
  • a preferred example of a method for complexity reduction includes for example AFLP® (Keygene N. V., the Netherlands; see e.g. EP 0 534 858, US6045994) , the methods described by Dong (see e.g.
  • the complexity reduction methods used in the present invention have in common that they are reproducible. Reproducible in the sense that when the same sample is reduced in complexity in the same manner, the same subset of the sample is obtained, as opposed to more random complexity reduction such as microdissection or the use of ⁇ iRNA (cDNA) which represents a portion of the genome transcribed in a selected tissue and for its reproducibility is depending on the selection of tissue, time of isolation etc.
  • AFLP refers to a method for selective amplification of DNA based on digesting a nucleic acid with one or more restriction endonucleases to yield restriction fragments, ligating adaptors to the restriction fragments and amplifying the adaptor-ligated restriction fragments with at least one primer that is (part) complementary to the adaptor, (part) complementary to the remains of the restriction endonuclease, and that further contains at least one randomly selected nucleotide from amongst A, C, T, or G (or U as the case may be) .
  • AFLP does not require any prior sequence information and can be performed on any starting DNA.
  • AFLP comprises the steps of:
  • AFLP thus provides a reproducible subset of adaptor-ligated fragments.
  • AFLP is described in EP 534858, US 6045994 and in Vos et al. Reference is made to these publications for further details regarding AFLP.
  • the AFLP is commonly used as a complexity reduction technique and a DNA fingerprinting technology. Within the context of the use of AFLP as a fingerprinting technology, the concept of an AFLP marker has been developed.
  • An AFLP marker is an amplified adaptor-ligated restriction fragment that is different between two samples that have been amplified using AFLP (fingerprinted) , using the same set of primers. As such, the presence or absence of this amplified adaptor- ligated restriction fragment can be used as a marker that is linked to a trait or phenotype .
  • an AFLP marker showes up as a band in the gel located at a certain mobility. Other electrophoretic techniques such as capillary electrophoresis may not refer to this as a band, but the concept remains the same, i.e. a nucleic acid with a certain length and mobility.
  • Absence or presence of the band may be indicative of (or associated with) the presence or absence of the phenotype.
  • AFLP markers typically involve SNPs in the restriction site of the endonuclease or the selective nucleotides. Occasionally, AFLP markers may involve indels in the restriction fragment.
  • SNP marker is a marker that is based on an identified single nucleotide polymorphism at a certain position. SNP markers can be located at identical positions to AFLP markers, but SNP markers can also be located in the restriction fragment itself. As such the genus SNP markers thus encompasses the species AFLP markers .
  • Constant band a constant band in the AFLP technology is an amplified adaptor-ligated restriction fragment that is relatively invariable between samples.
  • a constant band in the AFLP technology will, over a range of samples, show up at about the same position in the gel, i.e. has the same length/mobility.
  • these are typically used to anchor the lanes corresponding to samples on a gel or electropherograms of multiple AFLP samples detected by capillary electrophoresis.
  • a constant band is less informative than an AFLP marker. Nevertheless, as AFLP markers customary involve SNPs in the selective nucleotides or the restriction site, constant bands may comprise SNPs in the restriction fragments themselves, rendering the constant bands an interesting alternative source of genetic information that is complementary to AFLP markers .
  • Selective base Located at the 3' end of the primer that contains a part that is complementary to the adaptor and a part that is complementary to the remains of the restriction site, the selective base is randomly selected from amongst A, C, T or G.
  • the subsequent amplification will yield only a reproducible subset of the adaptor- ligated restriction fragments, i.e. only the fragments that can be amplified using the primer carrying the selective base.
  • Selective nucleotides can be added to the 3' end of the primer in a number varying between 1 and 10. Typically 1-4 suffice. Both primers may contain a varying number of selective bases.
  • the subset With each added selective base, the subset reduces the amount of amplified adaptor- ligated restriction fragments in the subset by a factor of about 4.
  • the number of selective bases used in AFLP is indicated by +N+M, wherein one primer carries N selective nucleotides and the other primers carries M selective nucleotides.
  • an Eco/Mse +1/+2 AFLP is shorthand for the digestion of the starting DNA with EcoRI and Msel, ligation of appropriate adaptors and amplification with one primer directed to the EcoRI restricted position carrying one selective base and the other primer directed to the Msel restricted site carrying 2 selective nucleotides.
  • Clustering is meant the comparison of two or more nucleotide sequences based on the presence of short or long stretches of identical or similar nucleotides.
  • Several methods for alignment of nucleotide sequences are known in the art, as will be further explained below.
  • the terms “assembly” or “alignment” are used as synonyms.
  • Typical examples are ZIP sequences, known in the art as commonly used tags for unique detection by hybridization (Iannone et al. Cytometry 39:131-140, 2000). Using such a tag, the origin of a PCR sample can be determined upon further processing. In the case of combining processed products originating from different nucleic acid samples, the different nucleic acid samples are generally identified using different tags. In the case of the present invention, the addition of a unique sequence tag serves to identify the coordinates of the individual plant in the pool of sequences amplification products. Multiple tags can be used. Tagging: the term tagging refers to the addition of a tag to a nucleic acid sample in order to be able to distinguish it from a second or further nucleic acid sample.
  • Tagging can e.g. be performed by the addition of a sequence identifier during complexity reduction or by any other means known in the art.
  • sequence identifier can e.g. be a unique base sequence of varying but defined length uniquely used for identifying a specific nucleic acid sample. Typical examples thereof are for instance ZIP sequences.
  • the origin of a sample can be determined upon further processing.
  • the different nucleic acid samples should be identified using different tags.
  • Tagged library refers to a library of tagged nucleic acids.
  • sequencing refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA.
  • High-throughput screening is a method for scientific experimentation especially relevant to the fields of biology and chemistry. Through a combination of modern robotics and other specialised laboratory hardware, it allows a researcher to effectively screen large amounts of samples simultaneously.
  • Restriction endonuclease a restriction endonuclease or restriction enzyme is an enzyme that recognizes a specific nucleotide sequence (target site) in a double-stranded DNA molecule, and will cleave both strands of the DNA molecule at every target site.
  • Restriction fragments the DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome (or nucleic acid, regardless of its origin) will be digested by a particular restriction endonuclease into a discrete set of restriction fragments.
  • the DNA fragments that result from restriction endonuclease cleavage can be further used in a variety of techniques and can for instance be detected by gel electrophoresis .
  • Gel electrophoresis in order to detect restriction fragments, an analytical method for fractionating double-stranded DNA molecules on the basis of size can be required. The most commonly used technique for achieving such fractionation is (capillary) gel electrophoresis.
  • the rate at which DNA fragments move in such gels depends on their molecular weight; thus, the distances travelled decrease as the fragment lengths increase.
  • the DNA fragments fractionated by gel electrophoresis can be visualized directly by a staining procedure e.g. silver staining or staining using ethidium bromide, if the number of fragments included in the pattern is sufficiently small.
  • further treatment of the DNA fragments may incorporate detectable labels in the fragments, such as fluorophores or radioactive labels.
  • Ligation the enzymatic reaction catalyzed by a ligase enzyme in which two double-stranded DNA molecules are covalently joined together is referred to as ligation.
  • both DNA strands are covalently joined together, but it is also possible to prevent the ligation of one of the two strands through chemical or enzymatic modification of one of the ends of the strands. In that case the covalent joining will occur in only one of the two DNA strands.
  • Synthetic oligonucleotide single-stranded DNA molecules having preferably from about 10 to about 50 bases, which can be synthesized chemically are referred to as synthetic oligonucleotides.
  • these synthetic DNA molecules are designed to have a unique or desired nucleotide sequence, although it is possible to synthesize families of molecules having related sequences and which have different nucleotide compositions at specific positions within the nucleotide sequence.
  • the term synthetic oligonucleotide will be used to refer to DNA molecules having a designed or desired nucleotide sequence .
  • Adaptors short double-stranded DNA molecules with a limited number of base pairs, e.g. about 10 to about 30 base pairs in length, which are designed such that they can be ligated to the ends of restriction fragments.
  • Adaptors are generally composed of two synthetic oligonucleotides which have nucleotide sequences which are partially complementary to each other.
  • the two synthetic oligonucleotides When mixing the two synthetic oligonucleotides in solution under appropriate conditions, they will anneal to each other forming a double-stranded structure. After annealing, one end of the adaptor molecule is designed such that it is compatible with the end of a restriction fragment and can be ligated thereto; the other end of the adaptor can be designed so that it cannot be ligated, but this need not be the case (double ligated adaptors) .
  • Adaptor-ligated restriction fragments restriction fragments that have been capped by adaptors .
  • Primers in general, the term primers refer to DNA strands which can prime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novo without primers: it can only extend an existing DNA strand in a reaction in which the complementary strand is used as a template to direct the order of nucleotides to be assembled.
  • PCR polymerase chain reaction
  • DNA amplification the term DNA amplification will be typically used to denote the in vitro synthesis of double-stranded DNA molecules using PCR. It is noted that other amplification methods exist and they may be used in the present invention without departing from the gist.
  • Selective hybridisation relates to hybridisation, under stringent hybridisation conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridisation to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
  • stringent conditions or “stringent hybridisation conditions” includes reference to conditions under which a probe will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background) . Stringent conditions are sequence-dependent and will be different in different circumstances.
  • target sequences can be identified which are 100% complementary to the probe (homologous probing) .
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) .
  • a probe is less than about 100 nucleotides in length, optionally no more than 50, or 25 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about is 30 0 C.
  • Stringent conditions may also be achieved with the addition of destabilising agents such as formamide.
  • destabilising agents such as formamide.
  • Exemplary moderate stringency conditions include hybridisation in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in 0.5* to 1*SSC at 55 to 60 0 C.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe. Tm is reduced by about 1 0 C. for each 1% of mismatching; thus, Tm, hybridisation and/or wash conditions can be adjusted to hybridise to sequences of the desired identity.
  • the Tm can be decreased 10 °C.
  • stringent conditions are selected to be about 5 0 C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilise a hybridisation and/or wash at 1, 2, 3, or 4 0 C. lower than the thermal melting point (Tm) ;
  • moderately stringent conditions can utilise a hybridisation and/or wash at 6, 7, 8, 9, or 10 0 C. lower than the thermal melting point (Tm) ;
  • low stringency conditions can utilise a hybridisation and/or wash at 11, 12, 13, 14, 15, or 20 0 C. lower than the thermal melting point (Tm) .
  • the present invention relates to a method for the high throughput discovery, detection and large-scale genotyping of one or more genetic markers in one or more samples, comprising the steps of:
  • the method relates to the discovery, detection and genotyping of one or more genetic markers in one or more samples. In certain embodiments, the methods relates to presence/absence scoring of the genetic markers of interest. In certain embodiments the method relates to determination of (co-) dominant genotypes of one more more samples for one or more genetic markers. This may require normalisation of the observed number of marker- or marker allele sequences between samples.
  • DNA is to be provided. This can be done by methods known in the art per se.
  • the isolation of DNA is generally achieved using common methods in the art such as the collection of tissue from a member of the population, DNA extraction (for instance using the Q-Biogene fast DNA kit) , quantification and normalisation to obtain equal amounts of DNA per sample.
  • the DNA can be from a variety of sources (Genomic, RNA, cDNA, BAc, YAC etc.) and organisms (human, mammal, plant, microorganisms, etc.).
  • the isolated DNA may be pooled.
  • the DNA is restricted in step (b) using at least one restriction endonuclease.
  • at least one restriction endonuclease Depending on the case, i.e. size of genome, more endonucleases can be used. In certain embodiments, 2 or more endonucleases can be used. For most genomes 2 endonucleases are sufficient and this is hence most preferred. In certain embodiments, especially for large or complex genomes, more endonucleases can be used. Preferably the endonuclease provides for relatively short restriction fragments in the order of 250-500 bp, but this is not essential. Typically, at least one frequent cutting endonuclease is preferred, i.e. endonucleases that have a 4 or 5 base pair recognition sequence.
  • One such enzyme is Msel, but numerous others are commercially available and can be used. Also enzymes that cut outside their recognition sequence can be used (Us type) , or enzymes that provide blunt ended restriction fragments. A preferred combination uses one rare (6 and more base pair recognition sequence, for example EcoRI) and one frequent cutter.
  • adaptors are ligated to the restriction fragments to provide for adaptor-ligated restriction fragments.
  • One or more different adaptors may be used, for instance two adaptors, one forward, one reverse adaptor.
  • one adaptor may be used for all fragments or sets of adaptors may be used that at the overhanging end of the adaptor contain permutations of nucleotides such as to provide for indexing linkers that may allow for a preselection step ( ⁇ nrau et al . , Gene, 1994, 145, 163-169).
  • blunt ended adaptors can be used, in the case of blunt ended restriction fragments.
  • Adaptor-ligation is well known in the art and is described inter alia in EP 534858.
  • One useful variant of the AFLP technology uses no selective nucleotides (i.e. +0/+0 primers) and is sometimes called linker PCR.
  • the selection step is provided by the use of restriction enzymes, different restriction enzymes yields different subsets.
  • This is sometimes also denoted as a pre-amplification wherein primers are used that are at least complementary to the adapters and optionally also to part of the remains of the recognition sequence of the restriction endonuclease.
  • Pre- amplification may serve to (further) normalize the amount of DNA from each sample, or to increase the total amount of DNA to allow for multiple analysis (i.e.
  • Pre-amplification may also be used to introduce tags that allow pooling prior to selective amplification.
  • tags for instance 4 bp
  • restriction fragments for a distinct sample can be tagged and at the end of the process can be retrieved by using the tag.
  • the adaptor-ligated restriction fragments are, after the optional pre-amplification, amplified in step (d) of the method of the invention with a pair of primers.
  • One of the primers is complementary to at least part of the adaptor and may further be complementary to part of the remainder of the recognition sequence of the endonuclease and may further contain (randomly selected) selective nucleotides at its 3' -end, similar as is described in EP534858.
  • the primers are capable of selectively hybridising under stringent hybridisation conditions.
  • the selective amplification can also be performed with primers that carry a 5' tag to identify the origin of the sample, similar as above.
  • the result is a library of (tagged) subsets of amplified adaptor-ligated restriction fragments.
  • the selectively amplified fragments in the libraries prepared from multiple samples can optionally be pooled at this point This may be useful in case markers are sought which are specific for certain groups of samples, such as those sharing certain phenotypic characteristics. Screening pooled samples is commonly referred to as bulked segregant analysis (BSA; Michelmore, Paran and Kesseli, 1991) . In certain embodiments, pooling can also be performed before DNA extraction in the sampling stage, reducing the number of DNA preparations. Pooling of the DNA further serves to normalise the DNAs prior to PCR amplification to provide for a more equal representation in the libraries for sequencing.
  • BSA bulked segregant analysis
  • The, optionally pooled, libraries of selectively amplified adaptor-ligated restriction fragments are now sequenced using high throughput sequencing technology.
  • the sequencing may in principle be conducted by any means known in the art, such as the dideoxy chain termination method (Sanger sequencing) . It is however preferred and more advantageous that the sequencing is performed using high-throughput sequencing methods, such as the methods disclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454 Life Sciences), by Seo et al . (2004) Proc. Natl. Acad. Sci. USA 101:5488-93, and technologies of Helios, Solexa, US Genomics, etcetera, which are herein incorporated by reference.
  • high-throughput sequencing methods such as the methods disclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454 Life Sciences), by Seo et al
  • sequencing is performed using the apparatus and/or method disclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454 Life Sciences) , which are herein incorporated by reference.
  • the technology described currently allows sequencing of up to 40 million bases in a single run and is 100 times faster and cheaper than competing technology based on Sanger sequencing and using currently available capillary electrophoresis instruments such as MegaBACE (GE Healthcare) or ABl3700(xl) (Applied Biosystems) . This will increase with increasing read length per reaction and/or increasing numbers of parallel reactions.
  • the sequencing technology roughly consists of 5 steps: 1) fragmentation of DNA and ligation of specific adaptor to create a library of single-stranded DNA (ssDNA) ; 2) annealing of ssDNA to beads, emulsification of the beads in water- in-oil microreactors and performing emulsion PCR to amplify the individual ssDNA molecules on beads; 3) selection of /enrichment for beads containing amplified ssDNA molecules on their surface 4) deposition of DNA carrying beads in a PicoTiterPlate®; and 5) simultaneous sequencing in 100,000 wells by generation of a pyrophosphate light signal.
  • the sequencing comprises the steps of: (1) annealing sequencing-adaptor-ligated fragments to beads, each bead annealing with a single fragment;
  • the adaptors that are present in the adaptor ligated restriction fragments are annealed to the beads.
  • the sequencing adaptor includes at least a "key" region for annealing to a bead, a sequencing primer region and a PCR primer region.
  • the amplified adaptor-ligated restriction fragments now contain at one of the ends the following sequence 5' -Sequence primer binding site Tag PCR primer sequence-
  • a segment is present that may be as follows: 5' -Bead annealing sequence Tag Adaptor specific sequence restriction site-specific sequence (optional) (randomly) selective sequence (optional)- 3'. It may be clear that the Sequence primer binding site and the Bead annealing sequence may be interchanged.
  • This Bead annealing sequence can now be used for annealing the fragments to the bead, the bead carrying a nucleotide sequence to that end.
  • adapted fragments are annealed to beads, each bead annealing with a single adapted fragment.
  • beads are added in excess as to ensure annealing of one single adapted fragment per bead for the majority of the beads (Poisson distribution) .
  • telomere sequence it is beneficial to amplify the PCR product directionally onto the bead for sequencing. This can be accomplished to perform the PCR with adaptor-tailed PCR primers of which one strand of the adaptor on the Msel (or other restriction enzyme) side is complementary to the oligonucleotide coupled to the sequence beads .
  • the beads are emulsified in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead.
  • PCR reagents are present in the water-in-oil microreactors allowing a PCR reaction to take place within the microreactors.
  • the microreactors are broken, and the beads comprising DNA (DNA positive beads) are enriched.
  • the beads are loaded in wells, each well comprising a single bead.
  • the wells are preferably part of a
  • PicoTiterTMPlate allowing for simultaneous sequencing of a large amount of fragments .
  • the sequence of the fragments is determined using pyrosequencing.
  • the PicoTiterTMPlate and the beads as well as the enzyme beads therein are subjected to different deoxyribonucleotides in the presence of conventional sequencing reagents, and upon incorporation of a deoxyribonucleotide a light signal is generated which is recorded. Incorporation of the correct nucleotide will generate a pyrosequencing signal which can be detected.
  • sequences of the fragments that are directly obtained from the sequencing step may be trimmed, preferably in silico, to remove any bead annealing sequence, sequencing primer, adaptor or primer-related sequence information.
  • the alignment or clustering is performed on sequence data that have been trimmed for any added adaptors/primer sequences i.e. using only the sequence data from the fragments that originate from the nucleic acid sample, together with the optional identifier tag.
  • NCBI Basic Local Alignment Search Tool (Altschul et al . , 1990) is available from several sources, including the National Center for Biological Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at ⁇ http://www.ncbi.nlm.nih.gov/BLAST/>. A description of how to determine sequence identity using this program is available at ⁇ http: //www.ncbi .nlm.nih.gov/BLAST/blast_help.html>.
  • the database preferably comprises EST sequences, genomic sequences of the species of interest and/or the non-redundant sequence database of GenBank or similar sequence databases.
  • High throughput sequencing methods can be used as described in Shendure et al. Science, VoI 309, Issue 5741, 1728-1732. Examples thereof are microelectrophoretic sequencing, Hybridization sequencing/sequencing by hybridization (SBH) , cyclic-array sequencing on amplified molecules, cyclic-array sequencing on single molecules, Non-cyclical, single-molecule, real-time methods, such as polymerase sequencing, exonuclease sequencing, nanopore sequencing.
  • SBH Hybridization sequencing/sequencing by hybridization
  • the method of the present invention can be used for the identification, detection of genotype determination AFLP markers, but also for the identification, detection and genotyping of SNP markers contained in constant bands .
  • the redundancy of the tagged amplified adaptor-ligated restriction fragments is at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9.
  • the sequence of each adaptor- ligated restriction fragment is determined at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9 fold.
  • the redundancy is selected such, assuming a 50/50 overall chance of identifying the locus correctly as homozygous, that the chance of correct identification of the locus is more than 95%, 96%, 97%, 98%, 99%, 99.5%.
  • the number of samples can be varied between 1 and 100.000, this also largely depends on the size of the genome to be analysed and the number of selectively amplified fragments. Usually, the capacity of the sequencing technology employed provides the most limiting factor in this respect.
  • Figure IA shows a fragment according to the present invention annealed onto a bead ( M54 bead' ) and the sequence of primer used for pre-amplification of the two pepper lines.
  • ⁇ DNA fragment' denotes the fragment obtained after digestion with a restriction endonuclease
  • ⁇ keygene adaptor' denotes an adaptor providing an annealing site for the (phosphorylated) oligonucleotide primers used to generate a library
  • ⁇ KRS' denotes an identifier sequence (tag)
  • Adaptor' denotes a sequencing adaptor
  • ⁇ 454 PCR adaptor' denotes an adaptor to allow for emulsion amplification of the DNA fragment.
  • FIG. 1B shows a schematic primer used in the complexity reduction step.
  • Such a primer generally comprises a recognition site region indicated as (2), a constant region that may include a tag section indicated as (1) and one or more selective nucleotides in a selective region indicated as (3) at the 3'-end thereof.).
  • Figure 2 shows DNA concentration estimation using 2% agarose gel-electrophoresis. Sl denotes PSPIl; S2 denote PI201234. 50, 100, 250 and 500 ng denotes respectively 50 ng, 100 ng, 250 ng and 500 ng to estimate DNA amounts of Sl and S2.
  • Fig 2C and 2D show DNA concentration determination using Nanodrop spectrophotometry.
  • Figure 3 shows the results of intermediate quality assessments of example 3.
  • Figure 4 shows flow charts of the sequence data processing pipeline, i.e. the steps taken from the generation of the sequencing data to the identification of putative SNPs, SSRs and indels, via steps of the removal of known sequence information in Trimming & Tagging resulting in trimmed sequence data which are clustered and assembled to yield contigs and singletons (fragments that cannot be assembled in a contig) after which putative polymorphisms can be identified and assessed.
  • Figure 4B further elaborates on the process of polymorphisms mining.
  • Figure 5 Multiple alignment "10037_CL989contig2" of pepper AFLP fragment sequences, containing a putative single nucleotide polymorphism (SNP) .
  • SNP single nucleotide polymorphism
  • PSPlI A allele present in both reads of sample 1
  • PI201234 G allele present in sample 2
  • Read names are shown on the left.
  • the consensus sequence of this multiple alignment is (5'- 3') : TAACACGACTTTGAACAAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACA[AZG] TGTTGGTTTT GGTGCTAACTTCAACCCCACTACTGTTTTGCTCTATTTTTG.
  • Figure 6 Graphic representation of the probability of correct classification of the genotype based on the number of observed reads per locus.
  • AFLP templates are prepared according to a modified protocol of Vos et al. which involves a heat-denaturation step for 20 min at
  • restriction enzyme digest is cooled to room temperature and DNA ligase is added.
  • the denaturation step leads to dissociation of the complementary strands of restriction fragments up to 120 bp such that no adaptors will be ligated to the ends. As a result, fragments smaller than 120 bp will not be amplified, hence size selection is achieved.
  • Pre-amplification reactions are performed as in conventional AFLP.
  • the last (selective) amplification step is performed using AFLP primers with unique identifier tags for every sample in the population/experiment, (using a unique 4 bp identifier sequence; KIS).
  • the KIS are located at the 5 ' end of the selective AFLP primers.
  • One additional selective nucleotide will be used in comparison with the number of selective bases used in conventional AFLP detection by electrophoresis, e.g. +4/+3 for an EcoRI/Msel fingerprint in pepper ⁇ (gel detection +3/+3) and +4/+4 for and EcoRI/Msel fingerprint in maize (gel detection +4/+3) .
  • the number of selective nucleotides that are applied needs to be determined empirically; it may be so that the same number of selective nucleotides can be applied as used for gel detection. This number further depends on the number of samples included in the experiment, since the numbers of sequence traces is assumed to be fixed 200,000 at the current status of sequencing technology, but this may and probably will increase. Preferred starting point is to achieve 10-fold sampling of AFLP fragments per sample library.
  • A) AFLP markers these are sequences which are observed in some samples, but absent in others. Inspection of the frequency of sequences in the collection of samples will reveal this category. Dominant scoring is performed depending on the presence/absence observation of these sequences in every sample. Reliable scoring of AFLP markers requires a statistical threshold to be set regarding the frequency with which other AFLP sequences are observed in the experiment. I.e. an AFLP marker can be scored as present (dominant) if the AFLP marker sequence is observed in the sample, but the reliability of the absent score depends on the (average) frequency of (constant) AFLP fragments. Statistical threshold levels are required such that presence/absence scoring is performed with preferably at least 99,5 % accuracy, depending on the acceptable level needed for the specific application. If a segregating population and its parents is analysed, these markers can possibly be scored co-dominantly as well by defining frequency categories of the marker sequences. The latter may actually be complicated by the influence of sampling variation of the AFLP marker which differs between samples.
  • SNP markers contained in the internal sequences of constant AFLP fragments are scored as co-dominant SNP markers. Again, this preferably requires applying a statistical threshold level for accurate calling of the presence or absence of an allele.
  • a 10-fold sequencing redundancy of the fragment library is expected to be sufficient but a statistical analysis method is needed to determine accuracy of the SNP marker genotypes depending on the number each allele sequence is observed.
  • the rationale is that when a constant band contains a SNP and one allele is observed e.g. 5 times while (the sequence containing the) other allele is not observed, it is highly likely that the sample is homozygous for the observed allele. Consequently, when both alleles are observed, the sample is scored heterozygous for the SNP marker, irrespective of their frequencies .
  • the result will be a genotyping table containing the genotypes of (co-) dominantly scored AFLP markers and co-dominantly scored SNPs, along with probabilities for correctness of the genotypes for all markers.
  • a dataset is generated which contains genotypes which have surpassed the set statistical threshold level.
  • the numbers of genetic markers observed depends on the SNP rate in the germplasm investigated. Below, estimates of the numbers of genetic markers are provided at different germplasm SNP rates, when sampling 20 kb sequence. The average length of AFLP markers/fragments is assumed to be 200 bp :
  • AFLP fragments may be sequenced from both ends, a proportion of the observed SNP can be derived from the same loci,
  • the numbers provided in table 1 are averages, which may differ between combinations of different primers. Analogous to conventional AFLP typing, identification of top primer combinations (PC) may yield higher numbers of markers per PC. In addition, the numbers presented in Table 1 may change depending on the required level of over sampling needed in order to reach the required accuracy level.
  • P(aa) is the fraction of the population with genotype aa (in the enclosed graph, fig 9, set at 0.25.
  • P(AA) is the fraction of the population with genotype AA (set at 0.25.
  • Example 1 PEPPER DNA from the Pepper lines PSP-Il and PI201234 was used to generate AFLP product by use of AFLP Keygene Recognition Site specific primers.
  • AFLP primers are essentially the same as conventional AFLP primers, e.g. described in EP 0 534 858, and will generally contain a recognition site region, a constant region and one or more selective nucleotides in a selective region.
  • the quality of the pre-amplification product of the two pepper samples was checked on a 1% agarose gel.
  • the preamplification products were 20 times diluted, followed by a KRSEcoRI +1(A) and KRSMseI +2 (CA) AFLP pre-amplification.
  • the KRS (identifier) sections are underlined and the selective nucleotides are in bold at the 3'- end in the primersequence SEQ ID 1-4 below.
  • the quality of the pre-amplification product of the two pepper samples was checked on a 1% agarose gel and by an EcoRI +3(A) and Msel +3 (C) (3) AFLP fingerprint (4) .
  • the pre-amplification products of the two pepper lines were separately purified on a QiagenPCR column (5) .
  • the concentration of the samples was measured on a NanoDrop ® ND-1000 Spectrophotometer.
  • a total of 5 micrograms PSP-Il and 5 micrograms PI201234 PCR products were mixed and sequenced.
  • Primer set I used for preamplification of PSP-Il
  • Msel-adaptor 92A18/92A19 5-GACGATGAGTCCTGAG-3 : 92A18 [SEQ ID 7]
  • PCR was performed in a PE GeneAmp PCR System 9700 and a 30 cycle profile was started with a 94°C denaturation step for 30 seconds, followed by an annealing step of 5 ⁇ °C for 60 seconds and an extension step of 72°C for 60 seconds.
  • AFLP protocol Selective amplification was done in a reaction volume of 20 ⁇ l.
  • the PCR was performed in a PE GeneAmp PCR System 9700.
  • a 13 cycle profile was started with a 94°C denaturation step for 30 seconds, followed by an annealing step of 65°C for 30 seconds, with a touchdown phase in witch the annealing temperature was lowered 0.7 0 C in each cycle, and an extension step of 72 0 C for 60 seconds.
  • This profile was followed by a 23 cycle profile with a 94 0 C denaturation step for 30 seconds, followed by an annealing step of 56°C for 30 seconds and an extension step of 72°C for 60 seconds.
  • the AFLP product was purified by using the QIAquick PCR Purification Kit (QIAGEN) following the QIAquick ® Spin Handbook 07/2002 page 18 and the concentration was measured with a NanoDrop ® ND-1000
  • the AFLP PCR products were first end-polished and subsequently ligated to adaptors to facilitate emulsion-PCR amplification and subsequent fragment sequencing as described by Margulies and co- workers.
  • 454 adaptor sequences, emulsion PCR primers, sequence- primers and sequence run conditions were all as described by Margulies and co-workers.
  • the linear order of functional elements in an emulsion-PCR fragment amplified on Sepharose beads in the 454 sequencing process was as follows as exemplified in figure IA:
  • raw 454 basecalled sequence reads were converted in FASTA format and inspected for the presence of tagged AFLP adaptor sequences using a BLAST algorithm.
  • sequences were trimmed, restriction endonuclease sites restored and assigned the appropriate tags (sample 1 EcoRI (ESl), sample 1 Msel (MSl), sample 2 EcoRI (ES2) or sample 2 Msel (MS2) , respectively) .
  • all trimmed sequences larger than 33 bases were clustered using a megaBLAST procedure based on overall sequence homologies.
  • clusters were assembled into one or more contigs and/or singletons per cluster, using a CAP3 multiple alignment algorithm.
  • Sequence mismatches were assigned quality scores based on the following criteria: * the numbers of reads in a contig * the observed allele distribution
  • Q scores range from 0 to 1; a Q score of 0.3 can only be reached in case both alleles are observed at least twice.
  • SNP polymorphism
  • SNP polymorphism
  • An elite polymorphism is thought to have a high probability of being located in a unique or low-copy genome sequence in case two homozygous lines have been used in the discovery process.
  • a weak association of a polymorphism with sample origin bears a high risk of having discovered false polymorphisms arising from alignment of non-allelic sequences in a contig.
  • DNA from the Maize lines B73 and M017 was used to generate AFLP product by use of AFLP Keygene Recognition Site specific primers.
  • AFLP primers are essentially the same as conventional AFLP primers, e.g. described in EP 0 534 858, and will generally contain a recognition site region, a constant region and one or more selective nucleotides at the 3' -end thereof.).
  • DNA from the pepper lines B73 or M017 was digested with the restriction endonucleases Taql (5U/reaction) for 1 hour at 65°C and Msel (2U/reaction) for 1 hour at 37°C following by inactivation for
  • restriction fragments were ligated with double-stranded synthetic oligonucleotide adapter, one end of witch is compatible with one or both of the ends of the Taql and/or Msel restriction fragments.
  • AFLP preamplification reactions (20 ⁇ l/reaction) with +1/+1 AFLP primers were performed on 10 times diluted restriction-ligation mixture.
  • PCR profile 20* (30 s at 94°C + 60 s at 56°C + 120 s at 72 0 C) .
  • Additional AFLP reactions 50 ⁇ l/reaction) with different +2 Taql and Msel AFLP Keygene Recognition Site primers (Table below, tags are in bold, selective nucleotides are underlined.) were performed on 20 times diluted +1/+1 Taql/Msel AFLP preamplification product.
  • PCR profile 30* (30 s at 94°C + 60 s at 56°C + 120 s at 72 0 C) .
  • the AFLP product was purified by using the QIAquick PCR Purification Kit (QIAGEN) following the QIAquick ® Spin Handbook 07/2002 page 18 and the concentration was measured with a NanoDrop ® ND-1000 Spectrophotometer. A total of 1.25 ⁇ g of each different B73 +2/+2 AFLP product and 1.25 ⁇ g of each different M017 +2/+2 AFLP product was put together and solved in 30 ⁇ l TE. Finally a mixture with a concentration of 333ng/ ⁇ l +2/4-2 AFLP product was obtained.
  • Pepper and maize AFLP fragment samples as prepared as described hereinbefore were processed by 454 Life Sciences as described (Margulies et al . , 2005. Genome sequencing in microfabricated high- density picolitre reactors. Nature 437 (7057) : 376-80. Epub July 31, 2005) .
  • KRS Recognition Sites
  • the resulting contigs from the assembly analysis form the basis of polymorphism detection.
  • Each ⁇ mismatch' in the alignment of each cluster is a potential polymorphism.
  • Selection criteria are defined to obtain a quality score:
  • SNPs and indels with a quality score above the threshold are identified as putative polymorphisms.
  • MISA MIcroSAtellite identification
  • This tool identifies di-, tri-, tetranucleotide and compound SSR motifs with predefined criteria and summarizes occurrences of these SSRs.
  • Example 3 SNP validation by PCR amplification and Sanger sequencing
  • STS sequence tagged site
  • Primer_1.2f 5'- AAACCCAAACTCCCCCAATC-3 ' , [SEQ ID 33] and Primer_1.2r:5 f - AGCGGATAACAATTTCACACAGGACATCAGTAGTCACACTGGTA
  • primer 1.2r contained an M13 sequence primer binding site and length stuffer at its 5 prime end.
  • PCR amplification was carried out using +A/+CA AFLP amplification products of PSPlI and PI210234 prepared as described in example 4 as template. PCR conditions were as follows: For 1 PCR reaction the following components were mixed:
  • PCR products were cloned into vector pCR2.1 (TA Cloning kit ; Invitrogen) using the TA Cloning method and transformed into INVaF' competent E. coli cells. Transformants were subjected to blue/white screening. Three independent white transformants each for PSPIl and PI-201234 were selected and grown 0/N in liquid selective medium for plasmid isolation. Plasmids were isolated using the QIAprep Spin Miniprep kit (QIAGEN) . Subsequently, the inserts of these plasmids were sequenced according to the protocol below and resolved on the MegaBACE 1000 (Amersham) . Obtained sequences were inspected on the presence of the SNP allele.
  • Two independent plasmids containing the PI-201234 insert and 1 plasmid containing the PSPIl insert contained the expected consensus sequence flanking the SNP. Sequence derived from the PSPlI fragment contained the expected A (underlined) allele and sequence derived from PI-201234 fragment contained the expected G allele (double underlined) :
  • PSPIl (sequence 1) : (5'-3 r )
  • PI-201234 (sequence 2) : (5' -3' ) AAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACAgTGTTGGTTTTGGTGCTAACTTCAA CCCCACTACTGTTTTGCTCTATTTTTG [SEQ ID 37]
  • AFLP a new technique for DNA fingerprinting. Nucl. Acids Res., 21, 4407-4414.

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Abstract

The invention relates to a method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of restriction endonuclease digest of DNA, adaptor-ligation, optional pre-amplif ication, selective amplification, pooling of the amplified products, sequencing the libraries with sufficient redundancy, clustering followed by identification of the genetic markers within the library and/or between libraries and determination of (co-) dominant genotypes of the genetic markers.

Description

METHOD FOR HIGH-THROUGHPUT AFLP-BASED POLYMORPHISM DETECTION.
Technical Field
The present invention relates to the fields of molecular biology and genetics. The invention relates to rapid discovery, detection and large-scale genotyping of polymorphisms in a nucleic acid sample or between samples. The identified polymorphisms may be used as genetic markers.
Background of the invention Exploration of genomic DNA has long been desired by the scientific, in particular medical, community. Genomic DNA holds the key to identification, diagnosis and treatment of diseases such as cancer and Alzheimer's disease. In addition to disease identification and treatment, exploration of genomic DNA may provide significant advantages in plant and animal breeding efforts, which may provide answers to food and nutrition problems in the world.
Many diseases are known to be associated with specific genetic components, in particular with polymorphisms in specific genes. The identification of polymorphisms in large samples such as genomes is at present a laborious and time-consuming task. However, such identification is of great value to areas such as biomedical research, developing pharmacy products, tissue typing, genotyping and population studies.
Markers, i.e. genetic markers, have been used for a very long time as a genetic typing method, i.e. to connect a phenotypic trait to the presence, absence or amount of a particular part of DNA (gene) . One of the most versatile genetic typing technologies is AFLP, already around for many years and widely applicable to any organism (for reviews see Savelkoul et al. J. Clin. Microbiol, 1999, 37(10), 3083-3091; Bensch et al. Molecular Ecology, 2005, 14, 2899- 2914)
The AFLP technology (Zabeau & Vos, 1993; Vos et al., 1995) has found widespread use in plant breeding and other field since its invention in the early nineties. This is due to several characteristics of AFLP, of which the most important is that no prior sequence information is needed to generate large numbers of genetic markers in a reproducible fashion. In addition, the principle of selective amplification, a cornerstone of AFLP, ensures that the number of amplified fragments can be brought in line with the resolution of the detection system, irrespective of genome size or origin.
Detection of AFLP fragments is commonly carried out by electrophoresis on slab-gels (Vos et al.r 1995) or capillary electrophoresis (van der Meulen et al., 2002) . The majority of AFLP markers scored in this way represent (single nucleotide) polymorphisms occurring either in the restriction enzyme recognition sites used for AFLP template preparation or their flanking nucleotides covered by selective AFLP primers. The remainder of the AFLP markers are insertion/deletion polymorphisms occurring in the internal sequences of the restriction fragments and a very small fraction on single nucleotide substitutions occurring in small restriction fragments (< approximately 100 bp) , which for these fragments cause reproducible mobility variations between both alleles; these AFLP markers can be scored co-dominantly without having to rely on band intensities.
In a typical AFLP fingerprint, the AFLP markers therefore constitute the minority of amplified fragments (less than 50 percent but often less than 20 percent) , while the remainder are commonly referred to as constant AFLP fragments. The latter are nevertheless useful in the gel scoring procedure as they serve as anchor points to calculate fragments mobilities of AFLP markers and aid in quantifying the markers for co-dominant scoring. Co-dominant scoring (scoring for homo- or heterozygosity) of AFLP markers currently is restricted to the context of fingerprinting a segregating population. In a panel of unrelated lines, only dominant scoring is possible.
Although the throughput of AFLP is very high due to high multiplexing levels in the amplification and detection steps, the rate limiting step is the resolving power of electrophoresis. Electrophoresis allows unique identification of the majority of amplified fragments based on the combination of restriction enzyme combinations (EC) , primer combinations (PC) and mobility, but ideally, the detection system should be capable of determining the entire sequence of the amplified fragments to capture all polymorphisms . Detection by sequencing instead of mobility determination will increase throughput because:
1) polymorphisms located in the internal sequences will be detected in most (or all) amplified fragments; this will increase the number of markers per PC considerably. 2) no loss of AFLP markers due to co-migration of AFLP markers and constant bands .
3) co-dominant scoring does not rely on quantification of band intensities and is independent of the relatedness of the individuals fingerprinted. So far, detection of AFLP markers/sequences by sequencing has not been economically feasible due to, among other limitations, cost limitations of Sanger dideoxy sequencing technology and other conventional sequencing technologies.
Accordingly, it is one of the goals of the present invention to provide for economically feasible methods for the detection of AFLP markers or other genetic markers such as SNP markers based on sequencing.
An important problem further associated with detection of a collection of AFLP or SNP containing fragments via sequencing for genotyping (i.e. diagnostic) purposes is that of sampling variation.
Specifically, this means that when a collection of fragments is analyzed and particular fragments are not observed, one has to make sure that this is not due to the fact that the fragments involved were not sampled at the detection step, although they are present in the fragment mixture, because this would lead to false-negative scoring of the marker. This limitation does not apply to detection by electrophoresis because position information on the gel is available. Accordingly, it is one of the further goals of the present invention provide a method that solves the problem of sample variation or at least reduces the errors caused by sample variation to an acceptable minimum.
Summary of the invention
The present inventors have found that sequencing is within reach for the detection of AFLP and SNP markers with the use of AFLP in certain adapted procedures for high throughput sequencing. The invention thus provides a method or strategy which combines the power and generic applicability of AFLP with certain high throughput sequencing technologies to establish a generically applicable polymorphism scoring system. In this strategy, the issue of sampling variation is also addressed to ensure genotyping with high accuracy and maximizing chances for datasets with minimal numbers of missing genotypes .
Definitions
In the following description and examples a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided, unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references are incorporated herein in their entirety by reference. Polymorphism: polymorphism refers to the presence of two or more variants of a nucleotide sequence in a population. A polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphism includes e.g. a simple sequence repeat (SSR) and a single nucleotide polymorphism (SNP) , which is a variation, occurring when a single nucleotide: adenine (A), thymine (T) , cytosine (C) or guanine (G) - is altered. A variation must generally occur in at least 1% of the population to be considered a SNP. SNPs make up e.g. 90% of all human genetic variations, and occur every 100 to 300 bases along the human genome. Two of every three SNPs substitute Cytosine (C) with Thymine (T) . Variations in the DNA sequences of e.g. humans or plants can affect how they handle diseases, bacteria, viruses, chemicals, drugs, etc.
Nucleic acid: a nucleic acid according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes) . The present invention contemplates any deoxyribonucleotide,. ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or, glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
Complexity reduction: the term complexity reduction is used to denote a method wherein the complexity of a nucleic acid sample, such as genomic DNA, is reduced by the generation of a subset of the sample. This subset can be representative for the whole (i.e. complex) sample and is preferably a reproducible subset. Reproducible means in this context that when the same sample is reduced in complexity using the same method, the same, or at least comparable, subset is obtained. The method used for complexity reduction may be any method for complexity reduction known in the art. A preferred example of a method for complexity reduction includes for example AFLP® (Keygene N. V., the Netherlands; see e.g. EP 0 534 858, US6045994) , the methods described by Dong (see e.g. WO 03/012118, WO 00/24939), indexed linking (Unrau et al., vide infra), linker-PCR (WO90/008821) , and SALSA-PCR (WO00/23620) Schouten et al) etc. The complexity reduction methods used in the present invention have in common that they are reproducible. Reproducible in the sense that when the same sample is reduced in complexity in the same manner, the same subset of the sample is obtained, as opposed to more random complexity reduction such as microdissection or the use of πiRNA (cDNA) which represents a portion of the genome transcribed in a selected tissue and for its reproducibility is depending on the selection of tissue, time of isolation etc. AFLP: AFLP refers to a method for selective amplification of DNA based on digesting a nucleic acid with one or more restriction endonucleases to yield restriction fragments, ligating adaptors to the restriction fragments and amplifying the adaptor-ligated restriction fragments with at least one primer that is (part) complementary to the adaptor, (part) complementary to the remains of the restriction endonuclease, and that further contains at least one randomly selected nucleotide from amongst A, C, T, or G (or U as the case may be) . AFLP does not require any prior sequence information and can be performed on any starting DNA. In general, AFLP comprises the steps of:
(a) digesting a nucleic acid, in particular a DNA or cDNA, with one or more specific restriction endonucleases, to fragment the DNA into a corresponding series of restriction fragments; (b) ligating the restriction fragments thus obtained with a double-stranded synthetic oligonucleotide adaptor, one end of which is compatible with one or both of the ends of the restriction fragments, to thereby produce adapter-ligated, preferably tagged, restriction fragments of the starting DNA; (c) contacting the adapter-ligated, preferably tagged, restriction fragments under hybridizing conditions with one or more oligonucleotide primers that contain selective nucleotides at their 3' -end;
(d) amplifying the adapter-ligated, preferably tagged, restriction fragment hybridised with the primers by PCR or a similar technique so as to cause further elongation of the hybridised primers along the restriction fragments of the starting DNA to which the primers hybridised; and
(e) detecting, identifying or recovering the amplified or elongated DNA fragment thus obtained.
AFLP thus provides a reproducible subset of adaptor-ligated fragments. AFLP is described in EP 534858, US 6045994 and in Vos et al. Reference is made to these publications for further details regarding AFLP. The AFLP is commonly used as a complexity reduction technique and a DNA fingerprinting technology. Within the context of the use of AFLP as a fingerprinting technology, the concept of an AFLP marker has been developed.
AFLP marker: An AFLP marker is an amplified adaptor-ligated restriction fragment that is different between two samples that have been amplified using AFLP (fingerprinted) , using the same set of primers. As such, the presence or absence of this amplified adaptor- ligated restriction fragment can be used as a marker that is linked to a trait or phenotype . In conventional gel technology, an AFLP marker showes up as a band in the gel located at a certain mobility. Other electrophoretic techniques such as capillary electrophoresis may not refer to this as a band, but the concept remains the same, i.e. a nucleic acid with a certain length and mobility. Absence or presence of the band may be indicative of (or associated with) the presence or absence of the phenotype. AFLP markers typically involve SNPs in the restriction site of the endonuclease or the selective nucleotides. Occasionally, AFLP markers may involve indels in the restriction fragment.
SNP marker: a SNP marker is a marker that is based on an identified single nucleotide polymorphism at a certain position. SNP markers can be located at identical positions to AFLP markers, but SNP markers can also be located in the restriction fragment itself. As such the genus SNP markers thus encompasses the species AFLP markers .
Constant band: a constant band in the AFLP technology is an amplified adaptor-ligated restriction fragment that is relatively invariable between samples. Thus, a constant band in the AFLP technology will, over a range of samples, show up at about the same position in the gel, i.e. has the same length/mobility. In conventional AFLP these are typically used to anchor the lanes corresponding to samples on a gel or electropherograms of multiple AFLP samples detected by capillary electrophoresis. Typically, a constant band is less informative than an AFLP marker. Nevertheless, as AFLP markers customary involve SNPs in the selective nucleotides or the restriction site, constant bands may comprise SNPs in the restriction fragments themselves, rendering the constant bands an interesting alternative source of genetic information that is complementary to AFLP markers .
Selective base: Located at the 3' end of the primer that contains a part that is complementary to the adaptor and a part that is complementary to the remains of the restriction site, the selective base is randomly selected from amongst A, C, T or G. By extending a primer with a selective base, the subsequent amplification will yield only a reproducible subset of the adaptor- ligated restriction fragments, i.e. only the fragments that can be amplified using the primer carrying the selective base. Selective nucleotides can be added to the 3' end of the primer in a number varying between 1 and 10. Typically 1-4 suffice. Both primers may contain a varying number of selective bases. With each added selective base, the subset reduces the amount of amplified adaptor- ligated restriction fragments in the subset by a factor of about 4. Typically, the number of selective bases used in AFLP is indicated by +N+M, wherein one primer carries N selective nucleotides and the other primers carries M selective nucleotides. Thus, an Eco/Mse +1/+2 AFLP is shorthand for the digestion of the starting DNA with EcoRI and Msel, ligation of appropriate adaptors and amplification with one primer directed to the EcoRI restricted position carrying one selective base and the other primer directed to the Msel restricted site carrying 2 selective nucleotides.
Clustering: with the term "clustering" is meant the comparison of two or more nucleotide sequences based on the presence of short or long stretches of identical or similar nucleotides. Several methods for alignment of nucleotide sequences are known in the art, as will be further explained below. Sometimes the terms "assembly" or "alignment" are used as synonyms. Tag: a short sequence that can be added to a primer or included in its sequence or otherwise used as label to provide a unique identifier. Such a sequence identifier can be a unique base sequence of varying but defined length uniquely used for identifying a specific nucleic acid sample. For instance 4 bp tags allow 4 (exp4) = 256 different tags. Typical examples are ZIP sequences, known in the art as commonly used tags for unique detection by hybridization (Iannone et al. Cytometry 39:131-140, 2000). Using such a tag, the origin of a PCR sample can be determined upon further processing. In the case of combining processed products originating from different nucleic acid samples, the different nucleic acid samples are generally identified using different tags. In the case of the present invention, the addition of a unique sequence tag serves to identify the coordinates of the individual plant in the pool of sequences amplification products. Multiple tags can be used. Tagging: the term tagging refers to the addition of a tag to a nucleic acid sample in order to be able to distinguish it from a second or further nucleic acid sample. Tagging can e.g. be performed by the addition of a sequence identifier during complexity reduction or by any other means known in the art. Such sequence identifier can e.g. be a unique base sequence of varying but defined length uniquely used for identifying a specific nucleic acid sample. Typical examples thereof are for instance ZIP sequences. Using such a tag, the origin of a sample can be determined upon further processing. In case of combining processed products originating from different nucleic acid samples, the different nucleic acid samples should be identified using different tags.
Tagged library: the term tagged library refers to a library of tagged nucleic acids.
Sequencing: The term sequencing refers to determining the order of nucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA.
High-throughput screening: High-throughput screening, often abbreviated as HTS, is a method for scientific experimentation especially relevant to the fields of biology and chemistry. Through a combination of modern robotics and other specialised laboratory hardware, it allows a researcher to effectively screen large amounts of samples simultaneously.
Restriction endonuclease: a restriction endonuclease or restriction enzyme is an enzyme that recognizes a specific nucleotide sequence (target site) in a double-stranded DNA molecule, and will cleave both strands of the DNA molecule at every target site.
Restriction fragments: the DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome (or nucleic acid, regardless of its origin) will be digested by a particular restriction endonuclease into a discrete set of restriction fragments. The DNA fragments that result from restriction endonuclease cleavage can be further used in a variety of techniques and can for instance be detected by gel electrophoresis . Gel electrophoresis: in order to detect restriction fragments, an analytical method for fractionating double-stranded DNA molecules on the basis of size can be required. The most commonly used technique for achieving such fractionation is (capillary) gel electrophoresis. The rate at which DNA fragments move in such gels depends on their molecular weight; thus, the distances travelled decrease as the fragment lengths increase. The DNA fragments fractionated by gel electrophoresis can be visualized directly by a staining procedure e.g. silver staining or staining using ethidium bromide, if the number of fragments included in the pattern is sufficiently small. Alternatively further treatment of the DNA fragments may incorporate detectable labels in the fragments, such as fluorophores or radioactive labels.
Ligation: the enzymatic reaction catalyzed by a ligase enzyme in which two double-stranded DNA molecules are covalently joined together is referred to as ligation. In general, both DNA strands are covalently joined together, but it is also possible to prevent the ligation of one of the two strands through chemical or enzymatic modification of one of the ends of the strands. In that case the covalent joining will occur in only one of the two DNA strands. Synthetic oligonucleotide: single-stranded DNA molecules having preferably from about 10 to about 50 bases, which can be synthesized chemically are referred to as synthetic oligonucleotides. In general, these synthetic DNA molecules are designed to have a unique or desired nucleotide sequence, although it is possible to synthesize families of molecules having related sequences and which have different nucleotide compositions at specific positions within the nucleotide sequence. The term synthetic oligonucleotide will be used to refer to DNA molecules having a designed or desired nucleotide sequence . Adaptors: short double-stranded DNA molecules with a limited number of base pairs, e.g. about 10 to about 30 base pairs in length, which are designed such that they can be ligated to the ends of restriction fragments. Adaptors are generally composed of two synthetic oligonucleotides which have nucleotide sequences which are partially complementary to each other. When mixing the two synthetic oligonucleotides in solution under appropriate conditions, they will anneal to each other forming a double-stranded structure. After annealing, one end of the adaptor molecule is designed such that it is compatible with the end of a restriction fragment and can be ligated thereto; the other end of the adaptor can be designed so that it cannot be ligated, but this need not be the case (double ligated adaptors) .
Adaptor-ligated restriction fragments: restriction fragments that have been capped by adaptors . Primers: in general, the term primers refer to DNA strands which can prime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novo without primers: it can only extend an existing DNA strand in a reaction in which the complementary strand is used as a template to direct the order of nucleotides to be assembled. We will refer to the synthetic oligonucleotide molecules which are used in a polymerase chain reaction (PCR) as primers.
DNA amplification: the term DNA amplification will be typically used to denote the in vitro synthesis of double-stranded DNA molecules using PCR. It is noted that other amplification methods exist and they may be used in the present invention without departing from the gist.
Selective hybridisation: relates to hybridisation, under stringent hybridisation conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridisation to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. The terms "stringent conditions" or "stringent hybridisation conditions" includes reference to conditions under which a probe will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background) . Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridisation and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing) . Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) . Generally, a probe is less than about 100 nucleotides in length, optionally no more than 50, or 25 nucleotides in length. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about is 30 0C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilising agents such as formamide. Exemplary low stringency conditions include hybridisation with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37 0C, and a wash in 1* to 2*SSC (20*SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55 °C. Exemplary moderate stringency conditions include hybridisation in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37 0C, and a wash in 0.5* to 1*SSC at 55 to 60 0C. Exemplary high stringency conditions include hybridisation in 50% formamide, 1 M NaCl, 1% SDS at 37 0C, and a wash in 0.1*SSC at 60 to 65 0C. Specificity is typically the function of post-hybridisation washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem. , 138:267-284 (1984): Tm=81.5 0C. +16.6 (log M) +0.41 (% GC) -0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridisation solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe. Tm is reduced by about 1 0C. for each 1% of mismatching; thus, Tm, hybridisation and/or wash conditions can be adjusted to hybridise to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 °C. Generally, stringent conditions are selected to be about 5 0C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilise a hybridisation and/or wash at 1, 2, 3, or 4 0C. lower than the thermal melting point (Tm) ; moderately stringent conditions can utilise a hybridisation and/or wash at 6, 7, 8, 9, or 10 0C. lower than the thermal melting point (Tm) ; low stringency conditions can utilise a hybridisation and/or wash at 11, 12, 13, 14, 15, or 20 0C. lower than the thermal melting point (Tm) . Using the equation, hybridisation and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridisation and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 0C. (aqueous solution) or 32 °C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridisation of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology- Hybridisation with Nucleic Acid Probes, Part 1, Chapter 2 "Overview of principles of hybridisation and the strategy of nucleic acid probe assays", Elsevier, N. Y. (1993) ; and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995) .
Detailed description of the invention In a first aspect the present invention relates to a method for the high throughput discovery, detection and large-scale genotyping of one or more genetic markers in one or more samples, comprising the steps of:
(a) providing DNA from one or more samples; (b) restricting the DNA with at least one restriction endonuclease to produce restriction fragments;
(c) ligating adaptors to the restriction fragments to produce adaptor-ligated restriction fragments;
(d) optionally, amplifying the adaptor-ligated restriction fragments with a primer pair that is at least complementary to the adaptors to produce pre-amplified adaptor-ligated restriction fragments;
(e) amplifying the (optionally pre-amplified) adaptor- ligated restriction fragments with a primer pair, wherein at least one of the primers contains an identifier tag at the 5' end of the primer to produce a library of tagged amplified subsets of adaptor-ligated restriction fragments for each sample;
(f) optionally, pooling the libraries derived from multiple samples;
(g) sequencing the libraries using high throughput sequencing technology;
(h) clustering the sequences per library, using the identifier tag; (i) identify genetic markers by comparing clustered sequences within a library and/or between the libraries .
(j) determine (co-) dominant genotypes of the genetic markers in the one or more libraries, preferably for all samples and for all identified markers. The method relates to the discovery, detection and genotyping of one or more genetic markers in one or more samples. In certain embodiments, the methods relates to presence/absence scoring of the genetic markers of interest. In certain embodiments the method relates to determination of (co-) dominant genotypes of one more more samples for one or more genetic markers. This may require normalisation of the observed number of marker- or marker allele sequences between samples.
In the first step (a) of the method, DNA is to be provided. This can be done by methods known in the art per se. The isolation of DNA is generally achieved using common methods in the art such as the collection of tissue from a member of the population, DNA extraction (for instance using the Q-Biogene fast DNA kit) , quantification and normalisation to obtain equal amounts of DNA per sample. The DNA can be from a variety of sources (Genomic, RNA, cDNA, BAc, YAC etc.) and organisms (human, mammal, plant, microorganisms, etc.). The isolated DNA may be pooled.
The DNA is restricted in step (b) using at least one restriction endonuclease. Depending on the case, i.e. size of genome, more endonucleases can be used. In certain embodiments, 2 or more endonucleases can be used. For most genomes 2 endonucleases are sufficient and this is hence most preferred. In certain embodiments, especially for large or complex genomes, more endonucleases can be used. Preferably the endonuclease provides for relatively short restriction fragments in the order of 250-500 bp, but this is not essential. Typically, at least one frequent cutting endonuclease is preferred, i.e. endonucleases that have a 4 or 5 base pair recognition sequence. One such enzyme is Msel, but numerous others are commercially available and can be used. Also enzymes that cut outside their recognition sequence can be used (Us type) , or enzymes that provide blunt ended restriction fragments. A preferred combination uses one rare (6 and more base pair recognition sequence, for example EcoRI) and one frequent cutter.
After restriction of the pooled DNAs, or simultaneously therewith, adaptors are ligated to the restriction fragments to provide for adaptor-ligated restriction fragments. One or more different adaptors may be used, for instance two adaptors, one forward, one reverse adaptor. Alternatively one adaptor may be used for all fragments or sets of adaptors may be used that at the overhanging end of the adaptor contain permutations of nucleotides such as to provide for indexing linkers that may allow for a preselection step (ϋnrau et al . , Gene, 1994, 145, 163-169). Alternatively, blunt ended adaptors can be used, in the case of blunt ended restriction fragments. Adaptor-ligation is well known in the art and is described inter alia in EP 534858. One useful variant of the AFLP technology uses no selective nucleotides (i.e. +0/+0 primers) and is sometimes called linker PCR. As with Salsa PCR , the selection step is provided by the use of restriction enzymes, different restriction enzymes yields different subsets. This is sometimes also denoted as a pre-amplification wherein primers are used that are at least complementary to the adapters and optionally also to part of the remains of the recognition sequence of the restriction endonuclease. Pre- amplification may serve to (further) normalize the amount of DNA from each sample, or to increase the total amount of DNA to allow for multiple analysis (i.e. splitting up samples) and to enhance the signal-to-noise ratio. Pre-amplification may also be used to introduce tags that allow pooling prior to selective amplification. By the introduction of nucleotide tags (for instance 4 bp) at the 5' end of the primer, restriction fragments for a distinct sample can be tagged and at the end of the process can be retrieved by using the tag.
The adaptor-ligated restriction fragments are, after the optional pre-amplification, amplified in step (d) of the method of the invention with a pair of primers. One of the primers is complementary to at least part of the adaptor and may further be complementary to part of the remainder of the recognition sequence of the endonuclease and may further contain (randomly selected) selective nucleotides at its 3' -end, similar as is described in EP534858. Preferably the primers are capable of selectively hybridising under stringent hybridisation conditions. The selective amplification can also be performed with primers that carry a 5' tag to identify the origin of the sample, similar as above. The result is a library of (tagged) subsets of amplified adaptor-ligated restriction fragments. The selectively amplified fragments in the libraries prepared from multiple samples can optionally be pooled at this point This may be useful in case markers are sought which are specific for certain groups of samples, such as those sharing certain phenotypic characteristics. Screening pooled samples is commonly referred to as bulked segregant analysis (BSA; Michelmore, Paran and Kesseli, 1991) . In certain embodiments, pooling can also be performed before DNA extraction in the sampling stage, reducing the number of DNA preparations. Pooling of the DNA further serves to normalise the DNAs prior to PCR amplification to provide for a more equal representation in the libraries for sequencing.
The, optionally pooled, libraries of selectively amplified adaptor-ligated restriction fragments are now sequenced using high throughput sequencing technology.
The sequencing may in principle be conducted by any means known in the art, such as the dideoxy chain termination method (Sanger sequencing) . It is however preferred and more advantageous that the sequencing is performed using high-throughput sequencing methods, such as the methods disclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454 Life Sciences), by Seo et al . (2004) Proc. Natl. Acad. Sci. USA 101:5488-93, and technologies of Helios, Solexa, US Genomics, etcetera, which are herein incorporated by reference. It is most preferred that sequencing is performed using the apparatus and/or method disclosed in WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454 Life Sciences) , which are herein incorporated by reference. The technology described currently allows sequencing of up to 40 million bases in a single run and is 100 times faster and cheaper than competing technology based on Sanger sequencing and using currently available capillary electrophoresis instruments such as MegaBACE (GE Healthcare) or ABl3700(xl) (Applied Biosystems) . This will increase with increasing read length per reaction and/or increasing numbers of parallel reactions. The sequencing technology roughly consists of 5 steps: 1) fragmentation of DNA and ligation of specific adaptor to create a library of single-stranded DNA (ssDNA) ; 2) annealing of ssDNA to beads, emulsification of the beads in water- in-oil microreactors and performing emulsion PCR to amplify the individual ssDNA molecules on beads; 3) selection of /enrichment for beads containing amplified ssDNA molecules on their surface 4) deposition of DNA carrying beads in a PicoTiterPlate®; and 5) simultaneous sequencing in 100,000 wells by generation of a pyrophosphate light signal.
In a preferred embodiment, the sequencing comprises the steps of: (1) annealing sequencing-adaptor-ligated fragments to beads, each bead annealing with a single fragment;
(2) emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead;
(3) performing emulsion PCR to amplify adaptor-ligated fragments on the surface of beads
(4) selecting / enriching beads containing amplified adaptor- ligated fragments;
(5) loading the beads in wells, each well comprising a single bead; and (6) generating a pyrophosphate signal.
In the first step (1) , the adaptors that are present in the adaptor ligated restriction fragments are annealed to the beads. As outlined herein before, the sequencing adaptor includes at least a "key" region for annealing to a bead, a sequencing primer region and a PCR primer region. In particular, the amplified adaptor-ligated restriction fragments now contain at one of the ends the following sequence 5' -Sequence primer binding site Tag PCR primer sequence-
3' , while at the other end a segment is present that may be as follows: 5' -Bead annealing sequence Tag Adaptor specific sequence restriction site-specific sequence (optional) (randomly) selective sequence (optional)- 3'. It may be clear that the Sequence primer binding site and the Bead annealing sequence may be interchanged. This Bead annealing sequence can now be used for annealing the fragments to the bead, the bead carrying a nucleotide sequence to that end. Thus, adapted fragments are annealed to beads, each bead annealing with a single adapted fragment. To the pool of adapted fragments, beads are added in excess as to ensure annealing of one single adapted fragment per bead for the majority of the beads (Poisson distribution) .
In a preferred embodiment, to increase the efficiency of the screening further, it is beneficial to amplify the PCR product directionally onto the bead for sequencing. This can be accomplished to perform the PCR with adaptor-tailed PCR primers of which one strand of the adaptor on the Msel (or other restriction enzyme) side is complementary to the oligonucleotide coupled to the sequence beads .
In a next step, the beads are emulsified in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead. PCR reagents are present in the water-in-oil microreactors allowing a PCR reaction to take place within the microreactors. Subsequently, the microreactors are broken, and the beads comprising DNA (DNA positive beads) are enriched.
In a following step, the beads are loaded in wells, each well comprising a single bead. The wells are preferably part of a
PicoTiter™Plate allowing for simultaneous sequencing of a large amount of fragments .
After addition of enzyme-carrying beads, the sequence of the fragments is determined using pyrosequencing. In successive steps, the PicoTiter™Plate and the beads as well as the enzyme beads therein are subjected to different deoxyribonucleotides in the presence of conventional sequencing reagents, and upon incorporation of a deoxyribonucleotide a light signal is generated which is recorded. Incorporation of the correct nucleotide will generate a pyrosequencing signal which can be detected.
Pyrosequencing itself is known in the art and described inter alia on www.biotagebio.com; www.pyrosequencing.com / section technology. The technology is further applied in e.g. WO 03/004690, WO 03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454 Life Sciences), which are herein incorporated by reference.
After sequencing, the sequences of the fragments that are directly obtained from the sequencing step may be trimmed, preferably in silico, to remove any bead annealing sequence, sequencing primer, adaptor or primer-related sequence information. Typically, the alignment or clustering is performed on sequence data that have been trimmed for any added adaptors/primer sequences i.e. using only the sequence data from the fragments that originate from the nucleic acid sample, together with the optional identifier tag.
Methods of alignment of sequences for comparison purposes are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman (1981) Adv. Appl . Math. 2:482; Needleman and Wunsch (1970) J. MoI. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucl. Acids Res. 16:10881-90; Huang et al . (1992) Computer Appl. in the Biosci. 8:155-65; and Pearson et al . (1994) Meth. MoI. Biol. 24:307-31, which are herein incorporated by reference . Altschul et al . (1994) Nature Genet. 6:119-29 (herein incorporated by reference) present a detailed consideration of sequence alignment methods and homology calculations .
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al . , 1990) is available from several sources, including the National Center for Biological Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at <http://www.ncbi.nlm.nih.gov/BLAST/>. A description of how to determine sequence identity using this program is available at <http: //www.ncbi .nlm.nih.gov/BLAST/blast_help.html>. The database preferably comprises EST sequences, genomic sequences of the species of interest and/or the non-redundant sequence database of GenBank or similar sequence databases.
High throughput sequencing methods can be used as described in Shendure et al. Science, VoI 309, Issue 5741, 1728-1732. Examples thereof are microelectrophoretic sequencing, Hybridization sequencing/sequencing by hybridization (SBH) , cyclic-array sequencing on amplified molecules, cyclic-array sequencing on single molecules, Non-cyclical, single-molecule, real-time methods, such as polymerase sequencing, exonuclease sequencing, nanopore sequencing.
Within the library the presence of a genetic marker and/or the genotype of the sample for a genetic marker can now be determined.
The method of the present invention can be used for the identification, detection of genotype determination AFLP markers, but also for the identification, detection and genotyping of SNP markers contained in constant bands .
To provide a solution to the problem of sampling variation which affects the accuracy of genotyping genetic markers by sequencing allelic (marker) fragments contained in a library of multiple fragments, the present inventors have also found that detection of AFLP markers via sequencing is preferably performed with sufficient redundancy (depth) to sample all amplified fragments at least once and accompanied by statistical means which address the issue of sampling variation in relation to the accuracy of the genotypes called. Furthermore, just as with AFLP scoring, in the context of a segregating population, the simultaneous scoring of the parent individuals in one experiment, will aid in determining the statistical threshold, because all possible alleles in the sample will be scored in either parent 1 or parent 2. Note that it is suggested to sample parent individuals with higher redundancy than individuals of segregating populations.
Thus, in certain embodiments, the redundancy of the tagged amplified adaptor-ligated restriction fragments is at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9. In certain embodiments, the sequence of each adaptor- ligated restriction fragment is determined at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9 fold. In certain embodiments, the redundancy is selected such, assuming a 50/50 overall chance of identifying the locus correctly as homozygous, that the chance of correct identification of the locus is more than 95%, 96%, 97%, 98%, 99%, 99.5%.
In certain embodiments, the number of samples can be varied between 1 and 100.000, this also largely depends on the size of the genome to be analysed and the number of selectively amplified fragments. Usually, the capacity of the sequencing technology employed provides the most limiting factor in this respect.
Brief description of the drawings
Figure IA shows a fragment according to the present invention annealed onto a bead ( M54 bead' ) and the sequence of primer used for pre-amplification of the two pepper lines. λDNA fragment' denotes the fragment obtained after digestion with a restriction endonuclease, Λkeygene adaptor' denotes an adaptor providing an annealing site for the (phosphorylated) oligonucleotide primers used to generate a library, λKRS' denotes an identifier sequence (tag) , M54 SEQ. Adaptor' denotes a sequencing adaptor, and λ454 PCR adaptor' denotes an adaptor to allow for emulsion amplification of the DNA fragment. The PCR adaptor allows for annealing to the bead and for amplification and may contain a 3'-T overhang. Figure IB shows a schematic primer used in the complexity reduction step. Such a primer generally comprises a recognition site region indicated as (2), a constant region that may include a tag section indicated as (1) and one or more selective nucleotides in a selective region indicated as (3) at the 3'-end thereof.). Figure 2 shows DNA concentration estimation using 2% agarose gel-electrophoresis. Sl denotes PSPIl; S2 denote PI201234. 50, 100, 250 and 500 ng denotes respectively 50 ng, 100 ng, 250 ng and 500 ng to estimate DNA amounts of Sl and S2. Fig 2C and 2D show DNA concentration determination using Nanodrop spectrophotometry. Figure 3 shows the results of intermediate quality assessments of example 3.
Figure 4 shows flow charts of the sequence data processing pipeline, i.e. the steps taken from the generation of the sequencing data to the identification of putative SNPs, SSRs and indels, via steps of the removal of known sequence information in Trimming & Tagging resulting in trimmed sequence data which are clustered and assembled to yield contigs and singletons (fragments that cannot be assembled in a contig) after which putative polymorphisms can be identified and assessed. Figure 4B further elaborates on the process of polymorphisms mining.
Figure 5: Multiple alignment "10037_CL989contig2" of pepper AFLP fragment sequences, containing a putative single nucleotide polymorphism (SNP) . Note that the SNP (indicated by an the black arrow) is defined by an A allele present in both reads of sample 1 (PSPlI) , denoted by the presence of the MSl tag in the name of the top two reads, and a G allele present in sample 2 (PI201234) , denoted by the presence of the MS2 tag in the name of the bottom two reads. Read names are shown on the left. The consensus sequence of this multiple alignment is (5'- 3') : TAACACGACTTTGAACAAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACA[AZG] TGTTGGTTTT GGTGCTAACTTCAACCCCACTACTGTTTTGCTCTATTTTTG.
Figure 6: Graphic representation of the probability of correct classification of the genotype based on the number of observed reads per locus. Examples
The method is exemplified as follows:
1) AFLP templates are prepared according to a modified protocol of Vos et al. which involves a heat-denaturation step for 20 min at
80 0C between the restriction and ligation steps. After incubation for 20 min at 80 0C, the restriction enzyme digest is cooled to room temperature and DNA ligase is added. The denaturation step leads to dissociation of the complementary strands of restriction fragments up to 120 bp such that no adaptors will be ligated to the ends. As a result, fragments smaller than 120 bp will not be amplified, hence size selection is achieved.
2) Pre-amplification reactions, if applicable, are performed as in conventional AFLP. 3) The last (selective) amplification step is performed using AFLP primers with unique identifier tags for every sample in the population/experiment, (using a unique 4 bp identifier sequence; KIS). The KIS are located at the 5 ' end of the selective AFLP primers. One additional selective nucleotide will be used in comparison with the number of selective bases used in conventional AFLP detection by electrophoresis, e.g. +4/+3 for an EcoRI/Msel fingerprint in pepper^ (gel detection +3/+3) and +4/+4 for and EcoRI/Msel fingerprint in maize (gel detection +4/+3) . The number of selective nucleotides that are applied needs to be determined empirically; it may be so that the same number of selective nucleotides can be applied as used for gel detection. This number further depends on the number of samples included in the experiment, since the numbers of sequence traces is assumed to be fixed 200,000 at the current status of sequencing technology, but this may and probably will increase. Preferred starting point is to achieve 10-fold sampling of AFLP fragments per sample library.
4) The collection of samples prepared according to steps 1-4 is subjected to sequencing via 454 Life Sciences technology. This means that individual AFLP fragments are cloned on beads, PCR amplified and sequenced. An output of 200,000 sequences of 100 bp length is expected. For a collection of 100 samples, this equals an average of 2000 sequences traces/sample, traceable to sample nr . via the 5' tag.
5) Assuming the amplification of 100 AFLP fragments per PC when 1 additional selective nucleotide is used compared to the number used with gel detection, of which 90 percent are constant bands, the AFLP fragments are sampled with 20-fold average redundancy per fragment. However, since sequencing is non directional and most bands are > 200 bp, sequencing redundancy will be slightly over 10-fold for each fragment end. 6) All sequences are clustered per sample using the KRS tag.
Given a 10-fold over sampling, this means that 200 different sequence traces are expected per sample, representing 200 x 100 bp = 20 kb sequence/sample. When 10 percent of these sequences are derived from AFLP markers (i.e. 1 allele is amplified and the other is absent in the PCR reaction) , 90 percent (18 kb) of the sequences are derived from constant bands.
7) Two types of genetic markers are scored:
A) AFLP markers : these are sequences which are observed in some samples, but absent in others. Inspection of the frequency of sequences in the collection of samples will reveal this category. Dominant scoring is performed depending on the presence/absence observation of these sequences in every sample. Reliable scoring of AFLP markers requires a statistical threshold to be set regarding the frequency with which other AFLP sequences are observed in the experiment. I.e. an AFLP marker can be scored as present (dominant) if the AFLP marker sequence is observed in the sample, but the reliability of the absent score depends on the (average) frequency of (constant) AFLP fragments. Statistical threshold levels are required such that presence/absence scoring is performed with preferably at least 99,5 % accuracy, depending on the acceptable level needed for the specific application. If a segregating population and its parents is analysed, these markers can possibly be scored co-dominantly as well by defining frequency categories of the marker sequences. The latter may actually be complicated by the influence of sampling variation of the AFLP marker which differs between samples.
B) (SN) polymorphisms in constant AFLP fragments.
This is the most interesting (and abundant) category of genetic markers. The essence is that SNP markers contained in the internal sequences of constant AFLP fragments are scored as co-dominant SNP markers. Again, this preferably requires applying a statistical threshold level for accurate calling of the presence or absence of an allele. A 10-fold sequencing redundancy of the fragment library is expected to be sufficient but a statistical analysis method is needed to determine accuracy of the SNP marker genotypes depending on the number each allele sequence is observed. The rationale is that when a constant band contains a SNP and one allele is observed e.g. 5 times while (the sequence containing the) other allele is not observed, it is highly likely that the sample is homozygous for the observed allele. Consequently, when both alleles are observed, the sample is scored heterozygous for the SNP marker, irrespective of their frequencies .
8) The result will be a genotyping table containing the genotypes of (co-) dominantly scored AFLP markers and co-dominantly scored SNPs, along with probabilities for correctness of the genotypes for all markers. Alternatively, a dataset is generated which contains genotypes which have surpassed the set statistical threshold level.
The approach assumes 10-fold over sampling of AFLP fragments per sample, yielding 18 kb of constant sequence/sample and 2 kb of AFLP marker sequences.
The numbers of genetic markers observed depends on the SNP rate in the germplasm investigated. Below, estimates of the numbers of genetic markers are provided at different germplasm SNP rates, when sampling 20 kb sequence. The average length of AFLP markers/fragments is assumed to be 200 bp :
Table 1. Expected numbers of genetic markers scored by sequencing AFLP fragments using
454 Life sciences technology assuming 10-fold over sampling,
200,000 sequence traces, 90 percent constant bands / 10 percent AFLP markers at various SNP rates.
Figure imgf000024_0001
* As the AFLP fragments may be sequenced from both ends, a proportion of the observed SNP can be derived from the same loci,
It is important to note that the numbers provided in table 1 are averages, which may differ between combinations of different primers. Analogous to conventional AFLP typing, identification of top primer combinations (PC) may yield higher numbers of markers per PC. In addition, the numbers presented in Table 1 may change depending on the required level of over sampling needed in order to reach the required accuracy level.
The calculation of the correct classification of the genotype is as follows:
P (correct) =P(aa) + P(AA) + P(Aa) * [1-0.5*exp (n-1) ) ]
Wherein P(aa) is the fraction of the population with genotype aa (in the enclosed graph, fig 9, set at 0.25. P(AA) is the fraction of the population with genotype AA (set at 0.25. P(Aa)Is the fraction of the population with genotype Aa (in fig 6 and table below, set at 0.5. n equals the number of individuals.
Table n P
1 0,5
2 0,75
3 0,875
4 0,9375
5 0,96875
6 0,984375
7 0,992188
8 0,996094
9 0,998047
10 0,999023
Example 1 PEPPER DNA from the Pepper lines PSP-Il and PI201234 was used to generate AFLP product by use of AFLP Keygene Recognition Site specific primers. (These AFLP primers are essentially the same as conventional AFLP primers, e.g. described in EP 0 534 858, and will generally contain a recognition site region, a constant region and one or more selective nucleotides in a selective region.
From the pepper lines PSP-Il or PI201234 150ng of DNA was digested with the restriction endonucleases EcoRI (5U/reaction) and Msel (2U/reaction) for 1 hour at 370C following by inactivation for 10 minutes at 800C. The obtained restriction fragments were ligated with double-stranded synthetic oligonucleotide adapter, one end of witch is compatible with one or both of the ends of the EcoRI and/or Msel restriction fragments. The restriction ligation mixture was 10 times diluted and 5 microliter of each sample was pre-amplified (2) with EcoRI +1(A) and Msel +1(C) primers (set I). After amplification the quality of the pre-amplification product of the two pepper samples was checked on a 1% agarose gel. The preamplification products were 20 times diluted, followed by a KRSEcoRI +1(A) and KRSMseI +2 (CA) AFLP pre-amplification. The KRS (identifier) sections are underlined and the selective nucleotides are in bold at the 3'- end in the primersequence SEQ ID 1-4 below. After amplification the quality of the pre-amplification product of the two pepper samples was checked on a 1% agarose gel and by an EcoRI +3(A) and Msel +3 (C) (3) AFLP fingerprint (4) . The pre-amplification products of the two pepper lines were separately purified on a QiagenPCR column (5) . The concentration of the samples was measured on a NanoDrop® ND-1000 Spectrophotometer. A total of 5 micrograms PSP-Il and 5 micrograms PI201234 PCR products were mixed and sequenced.
Primer set I used for preamplification of PSP-Il
EOlLKRSl 5 ' -CGTCAGACTGCGTACCAATTCA-3 ' [SEQ ID 1]
M15KKRS1 5 ' -TGGTGATGAGTCCTGAGTAACA-S ' [SEQ ID 2] Primer set II used for preamplification of PI201234 E01LKRS2 5 ' -CAAGAGACTGCGTACCAATTCA-3 ' [SEQ ID 3]
M15KKRS2 5 ' -AGCCGATGAGTCCTGAGTAACA-3 ' [SEQ ID 4]
(1) EcoRl/Msel restriction ligation mixture Restriction mix (40ul/sample)
DNA 6μl (+300ng)
ECoRI (5U) O.lμl
Msel (2U) 0.05μl
5xRL 8μl MQ 25.85μl
Totaal 40μl
Incubation during Ih. at 370C
Addition of:
Ligation mix (lOμl/sample) 1OmM ATP lμl
T4 DNA ligase Iμl
ECoRI adapt. (5pmol/μl) lμl Msel adapt. . (50pmol/μl) lμl
5xRL 2μl
MQ 4μl
Totaal lOμl Incubation during 3h. at 370C
EcoRI-adaptor
91M35/91M36: *-CTCGTAGACTGCGTACC : 91M35 [SEQ ID 5]
± bio CATCTGACGCATGGTTAΔ : 91M36 [SEQ ID 6]
Msel-adaptor 92A18/92A19: 5-GACGATGAGTCCTGAG-3 : 92A18 [SEQ ID 7]
3-TACTCAGGACTCAT-S :92A19 [SEQ ID 8]
(2) Pre-amplification
Preamplification (A/C) :
RL-mix (1Ox) 5μl EcoRI-pr EOlL (50ng/ul) 0.6μl
Msel-pr M02K (50ng/ul) 0.6μl dNTPs (25mM) O.lδμl
Taq.pol. (5U) 0.08μl
IOXPCR 2.0μl MQ 11.56μl
Total 20μl/reaction
Pre-amplification thermal profile
Selective pre amplification was done in a reaction volume of 50μl, The PCR was performed in a PE GeneAmp PCR System 9700 and a 20 cycle profile was started with a 94°C denaturation step for 30 seconds, followed by an annealing step of 56°C for 60 seconds and an extension step of 72°C for 60 seconds.
EcoRI +1(A)1
EO l L 92R11 : 5-AGACTGCGTACCAATTCA-3 [ SEQ I D 9 ] Msel +1 ( C ) 1
M02 k 93E42 : 5-GATGAGTCCTGAGTAAC-S [ SEQ ID 10 ]
Preamplif ication A/CA :
PA+l/+l-mix (2Ox) :5μl
EcoRI-pr :1.5μl
Msel-pr . :1.5μl dNTPs (25mM) :0.4μl
Taq.pol. (5U) :0.2μl
IOXPCR :5μl
MQ :36.3μl Total : 50μl
Selective pre amplification was done in a reaction volume of 50μl. The PCR was performed in a PE GeneAmp PCR System 9700 and a 30 cycle profile was started with a 94°C denaturation step for 30 seconds, followed by an annealing step of 5β°C for 60 seconds and an extension step of 72°C for 60 seconds.
(3) KRSKcoRI +1(A) and KRSMseI +2(CA)2
05F212 EOlLKRSl CGTCAGACTGCGTACCAATTCA -3' [SEQ ID 11]
05F213 E01LKRS2 CAAGAGACTGCGTΆCCAΆTTCA —3 ' [SEQ ID 12]
05F214 M15KKRS1 TGGTGATGAGTCCTGAGTAΆCA -3' [SEQ ID 13]
05F215 M15KKRS2 AGCCGATGAGTCCTGAGTAACA -3' [SEQ ID 14] selective nucleotides in bold and tags (KRS) underlined Sample PSPIl : E01LKRS1/M15KKRS1 Sample PI120234 : E01LKRS2/M15KKRS2
(4) AFLP protocol Selective amplification was done in a reaction volume of 20μl. The PCR was performed in a PE GeneAmp PCR System 9700. A 13 cycle profile was started with a 94°C denaturation step for 30 seconds, followed by an annealing step of 65°C for 30 seconds, with a touchdown phase in witch the annealing temperature was lowered 0.70C in each cycle, and an extension step of 720C for 60 seconds. This profile was followed by a 23 cycle profile with a 940C denaturation step for 30 seconds, followed by an annealing step of 56°C for 30 seconds and an extension step of 72°C for 60 seconds. EcoRI +3 (AAC) and Msel +3 (CAG) E32 92S02: 5-GACTGCGTACCAATTCAAC-3 [SEQ ID 15] M49 92G23: 5-GATGAGTCCTGAGTAACAG-S [SEQ ID 16]
(5) Qiagen column
The AFLP product was purified by using the QIAquick PCR Purification Kit (QIAGEN) following the QIAquick® Spin Handbook 07/2002 page 18 and the concentration was measured with a NanoDrop® ND-1000
Spectrophotometer. A total of 5μg of +1/+2 PSP-Il AFLP product and 5μg of +1/+2 PI201234 AFLP product was put together and solved in 23.3μl TE. Finally a mixture with a concentration of 430ng/μl +1/+2 AFLP product was obtained. Sequence library preparation and high-throughput sequencing Mixed amplification products from both pepper lines were subjected to high-throughput sequencing using 454 Life Sciences sequencing technology as described by Margulies et al . , (Margulies et al . , Nature 437, pp. 376-380 and Online Supplements). Specifically, the AFLP PCR products were first end-polished and subsequently ligated to adaptors to facilitate emulsion-PCR amplification and subsequent fragment sequencing as described by Margulies and co- workers. 454 adaptor sequences, emulsion PCR primers, sequence- primers and sequence run conditions were all as described by Margulies and co-workers. The linear order of functional elements in an emulsion-PCR fragment amplified on Sepharose beads in the 454 sequencing process was as follows as exemplified in figure IA:
454 PCR adaptor - 454 sequence adaptor - 4 bp AFLP primer tag 1 - AFLP primer sequence 1 including selective nucleotide (s) - AFLP fragment internal sequence - AFLP primer sequence 2 including selective nucleotide (s) , 4 bp AFLP primers tag 2 - 454 sequence adaptor - 454 PCR adaptor - Sepharose bead
Two high-throughput 454 sequence runs were performed by 454 Life Sciences (Branford, CT; United States of America) . 454 sequence run data-processing. Sequence data resulting from one 454 sequence run were processed using a bio-informatics pipeline (Keygene N.V.).
Specifically, raw 454 basecalled sequence reads were converted in FASTA format and inspected for the presence of tagged AFLP adaptor sequences using a BLAST algorithm. Upon high-confidence matches to the known tagged AFLP primer sequences, sequences were trimmed, restriction endonuclease sites restored and assigned the appropriate tags (sample 1 EcoRI (ESl), sample 1 Msel (MSl), sample 2 EcoRI (ES2) or sample 2 Msel (MS2) , respectively) . Next, all trimmed sequences larger than 33 bases were clustered using a megaBLAST procedure based on overall sequence homologies. Next, clusters were assembled into one or more contigs and/or singletons per cluster, using a CAP3 multiple alignment algorithm. Contigs containing more than one sequence were inspected for the sequence mismatches, representing putative polymorphisms . Sequence mismatches were assigned quality scores based on the following criteria: * the numbers of reads in a contig * the observed allele distribution
The above two criteria form the basis for the so called Q score assigned to each putative SNP/indel. Q scores range from 0 to 1; a Q score of 0.3 can only be reached in case both alleles are observed at least twice.
* location in homopolymers of a certain length (adjustable; default setting to avoid polymorphism located in homopolymers of 3 bases or longer) .
* number of contigs in cluster. * distance to nearest neighboring sequence mismatches
(adjustable; important for certain types of genotyping assays probing flanking sequences)
* the level of association of observed alleles with sample 1 or sample 2; in case of a consistent, perfect association between the alleles of a putative polymorphism and samples 1 and 2, the polymorphism (SNP) is indicated as an "elite" putative polymorphism (SNP) . An elite polymorphism is thought to have a high probability of being located in a unique or low-copy genome sequence in case two homozygous lines have been used in the discovery process. Conversely, a weak association of a polymorphism with sample origin bears a high risk of having discovered false polymorphisms arising from alignment of non-allelic sequences in a contig.
Sequences containing SSR motifs were identified using the MISA search tool (MIcroSAtellelite identification tool; available from http : //pgrc . ipk-gatersleben . de/misa/
Overall statistics of the run is shown in the Table below.
Table. Overall statistics of a 454 sequence run for SNP discovery in pepper.
Figure imgf000030_0001
Figure imgf000031_0001
* SNP / indel mining criteria were as follows:
No neighbouring polymorphisms with Q score larger than 0.1 within 12 bases on either side, not present in homopolymers of 3 or more bases. Mining criteria did not take into account consistent association with sample 1 and 2, i.e. the SNPs and indels are not necessarily elite putative SNPs/indels
An example of a multiple alignment containing an elite putative single nucleotide polymorphism is shown in Figure 5.
Example 2 : Maize
DNA from the Maize lines B73 and M017 was used to generate AFLP product by use of AFLP Keygene Recognition Site specific primers.
(These AFLP primers are essentially the same as conventional AFLP primers, e.g. described in EP 0 534 858, and will generally contain a recognition site region, a constant region and one or more selective nucleotides at the 3' -end thereof.).
DNA from the pepper lines B73 or M017 was digested with the restriction endonucleases Taql (5U/reaction) for 1 hour at 65°C and Msel (2U/reaction) for 1 hour at 37°C following by inactivation for
10 minutes at 800C. The obtained restriction fragments were ligated with double-stranded synthetic oligonucleotide adapter, one end of witch is compatible with one or both of the ends of the Taql and/or Msel restriction fragments.
AFLP preamplification reactions (20μl/reaction) with +1/+1 AFLP primers were performed on 10 times diluted restriction-ligation mixture. PCR profile: 20* (30 s at 94°C + 60 s at 56°C + 120 s at 720C) . Additional AFLP reactions (50μl/reaction) with different +2 Taql and Msel AFLP Keygene Recognition Site primers (Table below, tags are in bold, selective nucleotides are underlined.) were performed on 20 times diluted +1/+1 Taql/Msel AFLP preamplification product. PCR profile: 30* (30 s at 94°C + 60 s at 56°C + 120 s at 720C) . The AFLP product was purified by using the QIAquick PCR Purification Kit (QIAGEN) following the QIAquick® Spin Handbook 07/2002 page 18 and the concentration was measured with a NanoDrop® ND-1000 Spectrophotometer. A total of 1.25μg of each different B73 +2/+2 AFLP product and 1.25μg of each different M017 +2/+2 AFLP product was put together and solved in 30μl TE. Finally a mixture with a concentration of 333ng/μl +2/4-2 AFLP product was obtained.
Table
Figure imgf000032_0001
Finally the 4 Pl-samples and the 4 P2-samples were pooled and concentrated. A total amount of 25μl of DNA product and a final concentration of 400ng/ul (total of lOμg) was obtained. Intermediate quality assessments are given in Figure 3.
SEQUENCING BY 454
Pepper and maize AFLP fragment samples as prepared as described hereinbefore were processed by 454 Life Sciences as described (Margulies et al . , 2005. Genome sequencing in microfabricated high- density picolitre reactors. Nature 437 (7057) : 376-80. Epub July 31, 2005) .
DATA PROCESSING Processing Pipeline: Input data raw sequence data were received for each run:
- 200,000 - 400,000 reads
- base calling quality scores -Trimming and tagging These sequence data are analyzed for the presence of Keygene
Recognition Sites (KRS) at the beginning and end of the read. These KRS sequences consist of both AFLP-adaptor and sample label sequence and are specific for a certain AFLP primer combination on a certain sample. The KRS sequences are identified by BLAST and trimmed and the restriction sites are restored. Reads are marked with a tag for identification of the KRS origin. Trimmed sequences are selected on length (minimum of 33 nt) to participate in further processing. Clustering and assembly A MegaBlast analysis is performed on all size-selected, trimmed reads to obtain clusters of homologous sequences. Consecutively all clusters are assembled with CAP3 to result in assembled contigs . From both steps unique sequence reads are identified that do not match any other reads. These reads are marked as singletons. The processing pipeline carrying out the steps described herein before is shown in Figure 4A
Polymorphism mining and quality assessment
The resulting contigs from the assembly analysis form the basis of polymorphism detection. Each Λmismatch' in the alignment of each cluster is a potential polymorphism. Selection criteria are defined to obtain a quality score:
- number of reads per contig
- frequency of λalleles' per sample - occurrence of homopolymer sequence
- occurrence of neighbouring polymorphisms
SNPs and indels with a quality score above the threshold are identified as putative polymorphisms. For SSR mining we use the MISA (MIcroSAtellite identification) tool (http://pgrc.ipk- gatersleben.de/misa). This tool identifies di-, tri-, tetranucleotide and compound SSR motifs with predefined criteria and summarizes occurrences of these SSRs.
The polymorphism mining and quality assignment process is shown in Figure 4B
RESULTS
The table below summarizes the results of the combined analysis of sequences obtained from 2 454 sequence runs for the combined pepper samples and 2 runs for the combined maize samples.
Figure imgf000034_0001
* both with selection against neighboring SNPs, at least 12 bp flanking sequence and not occurring in homopolymer sequences larger than 3 nucleotides.
Example 3. SNP validation by PCR amplification and Sanger sequencing In order to validate the putative A/G SNP identified in example 1, a sequence tagged site (STS) assay for this SNP was designed using flanking PCR primers. PCR primer sequences were as follows:
Primer_1.2f : 5'- AAACCCAAACTCCCCCAATC-3 ' , [SEQ ID 33] and Primer_1.2r:5f- AGCGGATAACAATTTCACACAGGACATCAGTAGTCACACTGGTA
CAAAAATAGAGCAAAACAGTAGTG -3' [SEQ ID 34]
Note that primer 1.2r contained an M13 sequence primer binding site and length stuffer at its 5 prime end. PCR amplification was carried out using +A/+CA AFLP amplification products of PSPlI and PI210234 prepared as described in example 4 as template. PCR conditions were as follows: For 1 PCR reaction the following components were mixed:
5 μl 1/10 diluted AFLP mixture (app. 10 ng/μl) 5 μl lprαol/μl primer 1.2f (diluted directly from a 500 μM stock) 5 μl lpmol/μl primer 1.2r (diluted directly from a 500 μM stock)
5 μl PCR mix - 2 μl 10 x PCR buffer
- 1 μl 5 inM dNTPs - 1.5 μl 25 mM MgCl2
- 0.5 μl H2O
5 μl Enzyme mix - 0.5 μl 10 x PCR buffer (Applied Biosystems)
- 0.1 μl 5U/μl AmpliTaq DNA polymerase (Applied Biosystems)
- 4.4 μl H2O The following PCR profile was used:
Cycle 1 2'; 94° C
Cycle 2-34 20' '; 94 C 3 30011 ••;; 5 566° C
2'30' r 72 C
Cycle 35 7'; 72° C
«>; 4° C
PCR products were cloned into vector pCR2.1 (TA Cloning kit ; Invitrogen) using the TA Cloning method and transformed into INVaF' competent E. coli cells. Transformants were subjected to blue/white screening. Three independent white transformants each for PSPIl and PI-201234 were selected and grown 0/N in liquid selective medium for plasmid isolation. Plasmids were isolated using the QIAprep Spin Miniprep kit (QIAGEN) . Subsequently, the inserts of these plasmids were sequenced according to the protocol below and resolved on the MegaBACE 1000 (Amersham) . Obtained sequences were inspected on the presence of the SNP allele. Two independent plasmids containing the PI-201234 insert and 1 plasmid containing the PSPIl insert contained the expected consensus sequence flanking the SNP. Sequence derived from the PSPlI fragment contained the expected A (underlined) allele and sequence derived from PI-201234 fragment contained the expected G allele (double underlined) :
PSPIl (sequence 1) : (5'-3r)
AAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACAATGTTGGTTTTGGTGCTAACTTCAA CCCCACTACTGTTTTGCTCTATTTTTGT [SEQ ID 35]
PI-201234 (sequence 1) : (5r -3' )
AAACCCAAACTCCCCCAATCGATTTCAAΆCCTAGAACAGTGTTGGTTTTGGTGCTΆACTTCAΆ CCCCACTACTGTTTTGCTCTATTTTTG [SEQ ID 36]
PI-201234 (sequence 2) : (5' -3' ) AAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACAgTGTTGGTTTTGGTGCTAACTTCAA CCCCACTACTGTTTTGCTCTATTTTTG [SEQ ID 37]
This result indicates that the putative pepper A/G SNP represents a true genetic polymorphism detectable using the designed STS assay.
REFERENCES
l.Zabeau,M. and Vos,P. (1993) Selective restriction fragment amplification; a general method for DNA fingerprinting. EP 0534858- Al, Bl, B2; US patent 6045994.
2.Vos,P., Hogers,R., Bleeker,M., Reijans,M., van de Lee, T., Homes, M., Frijters,A., Pot, J., Pelentan, J., Kuiper,M. et al. (1995) AFLP: a new technique for DNA fingerprinting. Nucl. Acids Res., 21, 4407-4414.
3. M. van der Meulen, J. Buntjer, M. J. T. van Eijk, P. Vos, and R. van Schaik. (2002) . Highly automated AFLP® fingerprint analysis on the MegaBACE capillary sequencer. Plant, Animal and Microbial Genome Xr San Diego, CA, January 12-16, P228, pp. 135. 4. Margulies et al., 2005. Genome sequencing in microfabricated high-density picolitre reactions. Nature advanced online publication 03959, August 1.
5. R. W. Michelmore, I. Paran, and R. V. Kesseli.
(1991) . Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci USA 88 (21) : 9828-32.
6. Shendure et al., 2005. Accurate multiplex polony sequencing of an evolved bacterial genome. Scienceexpress Report, August 4.

Claims

Claims
1. Method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of:
(a) providing DNA from one or more samples; (b) restricting the DNA with at least one restriction endonuclease to produce restriction fragments;
(c) ligating adaptors to the restriction fragments to produce adaptor-ligated restriction fragments;
(d) optionally, amplifying the adaptor-ligated restriction fragments with a primer pair that is complementary to the adaptors to produce pre-amplified adaptor-ligated restriction fragments;
(e) amplifying the (optionally pre-amplified) adaptor- ligated restriction fragments with a primer pair, wherein at least one of the primers contains an identifier tag at the 5' end of the primer to produce a library of tagged amplified subsets of adaptor-ligated restriction fragments for each sample;
(f) optionally, pooling the libraries; (g) sequencing the libraries using high throughput sequencing technology;
(h) clustering the sequences per library, using the identifier tag;
(i) identify genetic markers within the library and/or between libraries
(j) determine (co-) dominant genotypes of the genetic markers in one or more libraries.
2. Method according to claim 1, wherein the genetic marker is an AFLP marker or an SNP marker.
3. Method according to claim 1 or 2, wherein sequencing is based on sequencing by synthesis, preferably pyrosequencing.
4. Method according to claims 1-3, wherein sequencing is performed on a solid support such as a bead.
5. Method according to claims 1-4, wherein the sequencing comprises the steps of:
- annealing amplified adaptor-ligated restriction fragments to beads, each bead annealing with a single adaptor-ligated fragment; - emulsifying the beads in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead;
- performing emulsion PCR to amplify the adaptor -ligated restriction fragments on the surface of the beads; - loading the beads in wells, each well comprising a single bead; and
- generating a pyrophosphate signal.
6. Method according to claims 1-4, wherein the average redundancy of the tagged amplified adaptor-ligated restriction fragments is at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9.
7. Method according to claims 1-5, wherein the sequence of each adaptor-ligated restriction fragment is determined at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9 fold.
8. Method according to claims 1-6, wherein between endonuclease restriction and adaptor ligation a size selection is performed by a denaturation step.
9. Method according to claims 1-8, wherein the DNA is selected from the group consisting of genomic DNA, RNA, cDNA, BACs,
YACs, whole-genome amplified DNA, PCR product.
10. Method according to claims 1-9, wherein the adaptor is a double stranded synthetic oligonucleotide adaptor having one end that is compatible with one or both ends of the restriction fragments.
11. Method according to claims 1-10, wherein the DNA is restricted with two, preferably three, or more restriction endonucleases .
12. Method according to claims 1-11, wherein the DNA is restricted with two restriction endonucleases.
13. Method according to claims 1-2, wherein at least one of the restriction endonucleases is a rare cutter.'
14. Method according to claims 1-13, wherein at least one of the restriction endonucleases is a frequent cutter.
15. Method according to claims 1-14, wherein the primer contains from one to 10 (preferably randomly selected from amongst A, C, T or G) selective nucleotides, more preferably from one to 5 nucleotides .
16. Method according to any of the previous claims wherein the DNA is restricted using a combination of three or more restriction endonucleases .
17. Use of the method as defined in any of the above claims for co- dominant scoring of AFLP and/or SNP marker sequences.
18. Use of high throughput sequencing methodology for the detection of polymorphisms in of the method as defined in any of the above method claims, for genotyping purposes including genetic mapping, QTL mapping, fine mapping genes/traits, linkage disequilibrium (LD) mapping, marker-assisted back-crossing, genetic distance analysis, discovery of markers linked to traits or phenotypes, diagnostic genotyping of patient samples etc.
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Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009068604A1 (en) * 2007-11-27 2009-06-04 Vib Vzw Marker assisted directed evolution
EP2172565A1 (en) * 2008-09-24 2010-04-07 Cyano Biotech GmbH Method of identifying and/or differentiating cyanophyta
WO2010064897A1 (en) 2008-12-05 2010-06-10 Keygene N.V. Farmesene synthase
WO2011074960A1 (en) * 2009-12-17 2011-06-23 Keygene N.V. Restriction enzyme based whole genome sequencing
WO2012096579A2 (en) 2011-01-14 2012-07-19 Keygene N.V. Paired end random sequence based genotyping
CN102864498A (en) * 2012-09-24 2013-01-09 天津工业生物技术研究所 Establishment method of long mate pair library
KR20130038353A (en) * 2010-06-30 2013-04-17 비지아이 션전 코포레이션 리미티드 New pcr sequencing method and use thereof in hla genotyping
US20140221217A1 (en) * 2011-07-08 2014-08-07 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
EP2802666A1 (en) 2012-01-13 2014-11-19 Data2Bio Genotyping by next-generation sequencing
WO2015083004A1 (en) * 2013-12-02 2015-06-11 Population Genetics Technologies Ltd. Method for evaluating minority variants in a sample
EP3187040A1 (en) 2015-12-30 2017-07-05 Vilmorin et Cie Resistance to tolcndv in melons
AU2013326406B2 (en) * 2012-10-05 2019-01-03 Katholieke Universiteit Leuven, KU LEUVEN R&D High-throughput genotyping by sequencing low amounts of genetic material
KR20190019049A (en) * 2016-04-15 2019-02-26 메나리니 실리콘 바이오시스템스 에스.피.에이. METHOD AND APPARATUS FOR GENERATING DNA LIBRARIES FOR LARGE PARALLEL SEQUENCES
US10266884B2 (en) 2009-04-30 2019-04-23 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US10308982B2 (en) 2010-04-05 2019-06-04 Prognosys Biosciences, Inc. Spatially encoded biological assays
WO2019121603A1 (en) 2017-12-18 2019-06-27 Keygene N.V. Chemical mutagenesis of cassava
US10450606B2 (en) 2012-02-17 2019-10-22 Fred Hutchinson Cancer Research Center Compositions and methods for accurately identifying mutations
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
US10619206B2 (en) 2013-03-15 2020-04-14 Tecan Genomics Sequential sequencing
WO2020109412A1 (en) 2018-11-28 2020-06-04 Keygene N.V. Targeted enrichment by endonuclease protection
WO2020169830A1 (en) 2019-02-21 2020-08-27 Keygene N.V. Genotyping of polyploids
US10774374B2 (en) 2015-04-10 2020-09-15 Spatial Transcriptomics AB and Illumina, Inc. Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US10774372B2 (en) 2013-06-25 2020-09-15 Prognosy s Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
WO2020243678A1 (en) * 2019-05-31 2020-12-03 North Carolina State University Compositions and methods related to quantitative reduced representation sequencing
WO2021116371A1 (en) 2019-12-12 2021-06-17 Keygene N.V. Semi-solid state nucleic acid manipulation
WO2021123062A1 (en) 2019-12-20 2021-06-24 Keygene N.V. Ngs library preparation using covalently closed nucleic acid molecule ends
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
WO2022074058A1 (en) 2020-10-06 2022-04-14 Keygene N.V. Targeted sequence addition
WO2022112394A1 (en) 2020-11-25 2022-06-02 Koninklijke Nederlandse Akademie Van Wetenschappen Ribosomal profiling in single cells
WO2022112316A1 (en) 2020-11-24 2022-06-02 Keygene N.V. Targeted enrichment using nanopore selective sequencing
US11352659B2 (en) 2011-04-13 2022-06-07 Spatial Transcriptomics Ab Methods of detecting analytes
US11584958B2 (en) 2017-03-31 2023-02-21 Grail, Llc Library preparation and use thereof for sequencing based error correction and/or variant identification
US11697843B2 (en) 2012-07-09 2023-07-11 Tecan Genomics, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US11733238B2 (en) 2010-04-05 2023-08-22 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20240084381A1 (en) * 2011-04-15 2024-03-14 The Johns Hopkins University Safe sequencing system
WO2024121354A1 (en) 2022-12-08 2024-06-13 Keygene N.V. Duplex sequencing with covalently closed dna ends
USRE50065E1 (en) 2012-10-17 2024-07-30 10X Genomics Sweden Ab Methods and product for optimising localised or spatial detection of gene expression in a tissue sample
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system
WO2024209000A1 (en) 2023-04-04 2024-10-10 Keygene N.V. Linkers for duplex sequencing

Families Citing this family (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105039313B (en) 2005-06-23 2018-10-23 科因股份有限公司 For the high throughput identification of polymorphism and the strategy of detection
DK1929039T4 (en) 2005-09-29 2014-02-17 Keygene Nv High throughput-screening af mutageniserede populationer
EP2789696B1 (en) 2005-12-22 2015-12-16 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
JP2009047956A (en) * 2007-08-21 2009-03-05 Sony Corp Imaging apparatus
EP2233580A1 (en) * 2009-03-05 2010-09-29 The Ohio State University Rapid genotyping of SNPs
CN101886132B (en) * 2009-07-15 2013-09-18 北京百迈客生物科技有限公司 Method for screening molecular markers correlative with properties based on sequencing technique and BSA (Bulked Segregant Analysis) technique
JP5799484B2 (en) * 2009-12-14 2015-10-28 トヨタ自動車株式会社 Probe design method in DNA microarray, DNA microarray having probe designed by the method
CN102061526B (en) * 2010-11-23 2014-04-30 深圳华大基因科技服务有限公司 DNA (deoxyribonucleic acid) library and preparation method thereof as well as method and device for detecting single nucleotide polymorphisms (SNPs)
EP2646573A1 (en) 2010-12-01 2013-10-09 MorphoSys AG Simultaneous detection of biomolecules in single cells
WO2012071685A1 (en) * 2010-12-02 2012-06-07 深圳华大基因科技有限公司 Method and system for bioinformatics analysis of hpv precise typing
CN102181943B (en) * 2011-03-02 2013-06-05 中山大学 Paired-end library construction method and method for sequencing genome by using library
WO2013097048A1 (en) * 2011-12-29 2013-07-04 深圳华大基因科技服务有限公司 Method and device for labelling single nucleotide polymorphism sites in genome
CN104508141A (en) * 2012-08-23 2015-04-08 深圳华大基因科技有限公司 Method and system for determining whether individual is in abnormal state
AU2013338393C1 (en) 2012-10-29 2024-07-25 The Johns Hopkins University Papanicolaou test for ovarian and endometrial cancers
CN104630202A (en) * 2013-11-13 2015-05-20 北京大学 Amplification method capable of decreasing bias generation during trace nucleic acid substance entire amplification
US11286531B2 (en) 2015-08-11 2022-03-29 The Johns Hopkins University Assaying ovarian cyst fluid
CN106676095A (en) * 2015-11-09 2017-05-17 中国科学院植物研究所 Complete set reagent for developing genetic markers and method for developing genetic markers through high-throughput sequencing
CN105695572B (en) * 2016-02-02 2021-02-23 中国水产科学研究院南海水产研究所 Method for developing molecular markers in large scale and efficiently based on Indel and SSR site technology
US10190155B2 (en) * 2016-10-14 2019-01-29 Nugen Technologies, Inc. Molecular tag attachment and transfer
CN106701949B (en) * 2016-12-30 2019-09-17 人和未来生物科技（长沙）有限公司 A kind of detection method of gene mutation and reagent reducing amplification bias
US11929145B2 (en) 2017-01-20 2024-03-12 Sequenom, Inc Methods for non-invasive assessment of genetic alterations
CN112236520A (en) 2018-04-02 2021-01-15 格里尔公司 Methylation signatures and target methylation probe plates
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
EP3856903A4 (en) 2018-09-27 2022-07-27 Grail, LLC Methylation markers and targeted methylation probe panel
WO2020123311A2 (en) 2018-12-10 2020-06-18 10X Genomics, Inc. Resolving spatial arrays using deconvolution
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
EP3973073A1 (en) * 2019-05-20 2022-03-30 Arima Genomics, Inc. Methods and compositions for enhanced genome coverage and preservation of spatial proximal contiguity
EP3976820A1 (en) 2019-05-30 2022-04-06 10X Genomics, Inc. Methods of detecting spatial heterogeneity of a biological sample
EP4025711A2 (en) 2019-11-08 2022-07-13 10X Genomics, Inc. Enhancing specificity of analyte binding
WO2021091611A1 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing
EP4081656A1 (en) 2019-12-23 2022-11-02 10X Genomics, Inc. Compositions and methods for using fixed biological samples in partition-based assays
FI3891300T3 (en) 2019-12-23 2023-05-10 10X Genomics Inc Methods for spatial analysis using rna-templated ligation
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
US12076701B2 (en) 2020-01-31 2024-09-03 10X Genomics, Inc. Capturing oligonucleotides in spatial transcriptomics
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US12110541B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Methods for preparing high-resolution spatial arrays
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US12129516B2 (en) 2020-02-07 2024-10-29 10X Genomics, Inc. Quantitative and automated permeabilization performance evaluation for spatial transcriptomics
US11835462B2 (en) 2020-02-11 2023-12-05 10X Genomics, Inc. Methods and compositions for partitioning a biological sample
US11211147B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Estimation of circulating tumor fraction using off-target reads of targeted-panel sequencing
US11475981B2 (en) 2020-02-18 2022-10-18 Tempus Labs, Inc. Methods and systems for dynamic variant thresholding in a liquid biopsy assay
US11211144B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Methods and systems for refining copy number variation in a liquid biopsy assay
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11926863B1 (en) 2020-02-27 2024-03-12 10X Genomics, Inc. Solid state single cell method for analyzing fixed biological cells
US11768175B1 (en) 2020-03-04 2023-09-26 10X Genomics, Inc. Electrophoretic methods for spatial analysis
ES2965354T3 (en) 2020-04-22 2024-04-12 10X Genomics Inc Methods for spatial analysis using targeted RNA deletion
EP4153775B1 (en) 2020-05-22 2024-07-24 10X Genomics, Inc. Simultaneous spatio-temporal measurement of gene expression and cellular activity
WO2021237087A1 (en) 2020-05-22 2021-11-25 10X Genomics, Inc. Spatial analysis to detect sequence variants
WO2021242834A1 (en) 2020-05-26 2021-12-02 10X Genomics, Inc. Method for resetting an array
CN116249785A (en) 2020-06-02 2023-06-09 10X基因组学有限公司 Space transcriptomics for antigen-receptor
WO2021247543A2 (en) 2020-06-02 2021-12-09 10X Genomics, Inc. Nucleic acid library methods
US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts
EP4421186A3 (en) 2020-06-08 2024-09-18 10X Genomics, Inc. Methods of determining a surgical margin and methods of use thereof
WO2021252591A1 (en) 2020-06-10 2021-12-16 10X Genomics, Inc. Methods for determining a location of an analyte in a biological sample
EP4172362B1 (en) 2020-06-25 2024-09-18 10X Genomics, Inc. Spatial analysis of dna methylation
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US11981960B1 (en) 2020-07-06 2024-05-14 10X Genomics, Inc. Spatial analysis utilizing degradable hydrogels
US11981958B1 (en) 2020-08-20 2024-05-14 10X Genomics, Inc. Methods for spatial analysis using DNA capture
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
WO2022140028A1 (en) 2020-12-21 2022-06-30 10X Genomics, Inc. Methods, compositions, and systems for capturing probes and/or barcodes
US20240076653A1 (en) * 2020-12-31 2024-03-07 Mokobio Life Science Corporation Beijing Method for constructing multiplex pcr library for high-throughput targeted sequencing
WO2022178267A2 (en) 2021-02-19 2022-08-25 10X Genomics, Inc. Modular assay support devices
WO2022198068A1 (en) 2021-03-18 2022-09-22 10X Genomics, Inc. Multiplex capture of gene and protein expression from a biological sample
EP4347879A1 (en) 2021-06-03 2024-04-10 10X Genomics, Inc. Methods, compositions, kits, and systems for enhancing analyte capture for spatial analysis
EP4196605A1 (en) 2021-09-01 2023-06-21 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
CN115131784B (en) * 2022-04-26 2023-04-18 东莞博奥木华基因科技有限公司 Image processing method and device, electronic equipment and storage medium

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0534858A1 (en) 1991-09-24 1993-03-31 Keygene N.V. Selective restriction fragment amplification : a general method for DNA fingerprinting
US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
WO2000024939A1 (en) * 1998-10-27 2000-05-04 Affymetrix, Inc. Complexity management and analysis of genomic dna
WO2000061800A2 (en) * 1999-04-09 2000-10-19 Keygene N.V. Method for the analysis of aflp® reaction mixtures using primer extension techniques
WO2005003375A2 (en) * 2003-01-29 2005-01-13 454 Corporation Methods of amplifying and sequencing nucleic acids
WO2006137733A1 (en) * 2005-06-23 2006-12-28 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
WO2006137734A1 (en) * 2005-06-23 2006-12-28 Keygene N.V. Improved strategies for sequencing complex genomes using high throughput sequencing technologies

Family Cites Families (89)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US24939A (en) 1859-08-02 Improvement in sewing-machines
EP0124990B1 (en) 1983-04-28 1987-08-19 Flexible Steel Lacing Company Method and apparatus for riveting fasteners to a belt
EP0456721A4 (en) 1989-01-31 1992-06-03 University Of Miami Microdissection and amplification of chromosomal dna
US20100267023A1 (en) 1992-09-24 2010-10-21 Keygene N.V. Selective restriction fragment amplification: fingerprinting
AU1682595A (en) * 1994-01-21 1995-08-08 North Carolina State University Methods for within family selection in woody perennials using genetic markers
EG23907A (en) 1994-08-01 2007-12-30 Delta & Pine Land Co Control of plant gene expression
WO1996017082A2 (en) * 1994-11-28 1996-06-06 E.I. Du Pont De Nemours And Company Compound microsatellite primers for the detection of genetic polymorphisms
US5565340A (en) * 1995-01-27 1996-10-15 Clontech Laboratories, Inc. Method for suppressing DNA fragment amplification during PCR
CA2286864A1 (en) * 1997-01-10 1998-07-16 Pioneer Hi-Bred International, Inc. Hybridization-based genetic amplification and analysis
US6090556A (en) * 1997-04-07 2000-07-18 Japan Science & Technology Corporation Method for quantitatively determining the expression of a gene
ES2163271T3 (en) 1997-05-13 2002-01-16 Azign Bioscience As PROCEDURE FOR CLONING ARNM AND VISUALIZING DIFFERENTIALLY EXPRESSED TRANSCRIPTORS (DODETS).
ATE369439T1 (en) 1997-12-15 2007-08-15 Csl Behring Gmbh MARKED PRIMER SUITABLE FOR DETECTION OF NUCLEIC ACIDS
CA2273616A1 (en) 1998-06-08 1999-12-08 The Board Of Trustees Of The Leland Stanford Junior University Method for parallel screening of allelic variation
US6124201A (en) 1998-06-12 2000-09-26 Advanced Micro Devices, Inc. Method for manufacturing semiconductors with self-aligning vias
ATE244771T1 (en) 1998-07-29 2003-07-15 Keygene Nv METHOD FOR DETECTING NUCLEIC ACID METHYLATIONS BY AFLP
US6232067B1 (en) 1998-08-17 2001-05-15 The Perkin-Elmer Corporation Adapter directed expression analysis
US6703228B1 (en) 1998-09-25 2004-03-09 Massachusetts Institute Of Technology Methods and products related to genotyping and DNA analysis
EP1001037A3 (en) 1998-09-28 2003-10-01 Whitehead Institute For Biomedical Research Pre-selection and isolation of single nucleotide polymorphisms
AU6372099A (en) 1998-10-16 2000-05-08 Keygene N.V. Method for the generation of dna fingerprints
US6480791B1 (en) 1998-10-28 2002-11-12 Michael P. Strathmann Parallel methods for genomic analysis
JP2002534098A (en) 1999-01-06 2002-10-15 コーネル リサーチ ファンデーション インク． Accelerated Identification of Single Nucleotide Polymorphisms and Alignment of Clones in Genome Sequencing
US20040029155A1 (en) * 1999-01-08 2004-02-12 Curagen Corporation Method for identifying a biomolecule
CA2358638A1 (en) 1999-01-12 2000-07-20 Quanam Medical Corporation Composition and methods for administration of water-insoluble paclitaxel derivatives
DE19911130A1 (en) 1999-03-12 2000-09-21 Hager Joerg Methods for identifying chromosomal regions and genes
AU778438B2 (en) 1999-04-06 2004-12-02 Yale University Fixed address analysis of sequence tags
US20040031072A1 (en) 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
US20020119448A1 (en) 1999-06-23 2002-08-29 Joseph A. Sorge Methods of enriching for and identifying polymorphisms
US20030204075A9 (en) * 1999-08-09 2003-10-30 The Snp Consortium Identification and mapping of single nucleotide polymorphisms in the human genome
AU7712400A (en) * 1999-09-23 2001-04-24 Gene Logic, Inc. Indexing populations
US6287778B1 (en) 1999-10-19 2001-09-11 Affymetrix, Inc. Allele detection using primer extension with sequence-coded identity tags
US6958225B2 (en) 1999-10-27 2005-10-25 Affymetrix, Inc. Complexity management of genomic DNA
AU1413601A (en) 1999-11-19 2001-06-04 Takara Bio Inc. Method of amplifying nucleic acids
AU2001253063A1 (en) 2000-03-31 2001-10-15 Fred Hutchinson Cancer Research Center Reverse genetic strategy for identifying functional mutations in genes of known sequence
US20110131679A2 (en) 2000-04-19 2011-06-02 Thomas La Rosa Rice Nucleic Acid Molecules and Other Molecules Associated with Plants and Uses Thereof for Plant Improvement
EP1282729A2 (en) * 2000-05-15 2003-02-12 Keygene N.V. Microsatellite-aflp
US7300751B2 (en) * 2000-06-30 2007-11-27 Syngenta Participations Ag Method for identification of genetic markers
CA2426824A1 (en) 2000-10-24 2002-07-25 The Board Of Trustees Of The Leland Stanford Junior University Direct multiplex characterization of genomic dna
US7141364B1 (en) * 2001-03-29 2006-11-28 Council Of Scientific And Industrial Research Universal primers for wildlife identification
US20040053236A1 (en) 2001-03-30 2004-03-18 Mccallum Claire M. Reverse genetic strategy for identifying functional mutations in genes of known sequences
WO2002083911A1 (en) 2001-04-12 2002-10-24 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Production of plants with increased tolerance to drought stress or with increased transpiration
WO2002086163A1 (en) 2001-04-20 2002-10-31 Karolinska Innovations Ab Methods for high throughput genome analysis using restriction site tagged microarrays
JP2005520484A (en) 2001-07-06 2005-07-14 ４５４ コーポレイション Method for isolating independent parallel chemical microreactions using a porous filter
WO2003020015A2 (en) 2001-08-30 2003-03-13 Purdue Research Foundation Methods to produce transgenic plants resistant to osmotic stress
EP1288301A1 (en) 2001-08-31 2003-03-05 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Berlin Plant-derived resistance gene
US6902921B2 (en) 2001-10-30 2005-06-07 454 Corporation Sulfurylase-luciferase fusion proteins and thermostable sulfurylase
US7504222B2 (en) 2001-10-31 2009-03-17 Millennium Pharmaceuticals, Inc. Compositions, kits, and methods for identification, assessment, prevention, and therapy of breast cancer
AU2002359436A1 (en) * 2001-11-13 2003-06-23 Rubicon Genomics Inc. Dna amplification and sequencing using dna molecules generated by random fragmentation
WO2003081988A2 (en) 2002-03-27 2003-10-09 Agrinomics Llc Generation of plants with improved drought tolerance
US6815167B2 (en) 2002-04-25 2004-11-09 Geneohm Sciences Amplification of DNA to produce single-stranded product of defined sequence and length
US20070065816A1 (en) * 2002-05-17 2007-03-22 Affymetrix, Inc. Methods for genotyping
US7108976B2 (en) * 2002-06-17 2006-09-19 Affymetrix, Inc. Complexity management of genomic DNA by locus specific amplification
WO2004001074A1 (en) 2002-06-21 2003-12-31 Lynx Therapeutics, Inc. Method for detecting foreign dna in a host genome
EP1546345B1 (en) 2002-09-05 2007-03-28 Plant Bioscience Limited Genome partitioning
US20040157238A1 (en) 2002-09-20 2004-08-12 Quinn John J. Method for detection of multiple nucleic acid sequence variations
WO2004035798A2 (en) 2002-10-18 2004-04-29 Cropdesign N.V. Identification of e2f target genes and uses thereof
EP1573009B1 (en) 2002-12-18 2011-09-21 Third Wave Technologies, Inc. Detection of small nucleic acids
JP2004208586A (en) 2002-12-27 2004-07-29 Wakunaga Pharmaceut Co Ltd Detection of hla(human leukocyte antigen)
EP1581661B1 (en) * 2003-01-10 2012-09-12 Keygene N.V. Aflp-based method for integrating physical and genetic maps
CA2555962C (en) * 2003-02-26 2015-10-06 Callida Genomics, Inc. Random array dna analysis by hybridization
JP4888876B2 (en) 2003-06-13 2012-02-29 田平 武 Recombinant adeno-associated virus vector for the treatment of Alzheimer's disease
ATE506442T1 (en) 2003-06-24 2011-05-15 Agrigenetics Inc PRODUCING PLANTS WITH IMPROVED DROUGHT TOLERANCE
WO2005002325A2 (en) 2003-06-24 2005-01-13 Agrinomics Llc Generation of plants with improved drought tolerance
US7051935B2 (en) 2003-07-28 2006-05-30 Imageid Ltd. Color calibration for color bar codes
WO2005026686A2 (en) 2003-09-09 2005-03-24 Compass Genetics, Llc Multiplexed analytical platform
US20050153317A1 (en) * 2003-10-24 2005-07-14 Metamorphix, Inc. Methods and systems for inferring traits to breed and manage non-beef livestock
EP3175914A1 (en) 2004-01-07 2017-06-07 Illumina Cambridge Limited Improvements in or relating to molecular arrays
US20050233354A1 (en) 2004-01-22 2005-10-20 Affymetrix, Inc. Genotyping degraded or mitochandrial DNA samples
AU2005214329A1 (en) 2004-02-12 2005-09-01 Population Genetics Technologies Ltd Genetic analysis by sequence-specific sorting
US7709262B2 (en) 2004-02-18 2010-05-04 Trustees Of Boston University Method for detecting and quantifying rare mutations/polymorphisms
EP1574585A1 (en) 2004-03-12 2005-09-14 Plant Research International B.V. Method for selective amplification of DNA fragments for genetic fingerprinting
JP4809594B2 (en) 2004-08-02 2011-11-09 東京エレクトロン株式会社 Inspection device
FR2876479B1 (en) 2004-10-11 2006-12-15 Parkeon PARTS MACHINE HAVING REMOVABLE CURRENCY COINS
US7220549B2 (en) 2004-12-30 2007-05-22 Helicos Biosciences Corporation Stabilizing a nucleic acid for nucleic acid sequencing
US7407757B2 (en) 2005-02-10 2008-08-05 Population Genetics Technologies Genetic analysis by sequence-specific sorting
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
EP1885882B1 (en) * 2005-05-10 2011-01-26 State of Oregon acting by & through the State Board of Higher Eduction on behalf of the University of Oregon Methods of mapping polymorphisms and polymorphism microarrays
EP2463386B1 (en) 2005-06-15 2017-04-12 Complete Genomics Inc. Nucleic acid analysis by random mixtures of non-overlapping fragments
US20070020640A1 (en) 2005-07-21 2007-01-25 Mccloskey Megan L Molecular encoding of nucleic acid templates for PCR and other forms of sequence analysis
DK1929039T4 (en) 2005-09-29 2014-02-17 Keygene Nv High throughput-screening af mutageniserede populationer
US10316364B2 (en) 2005-09-29 2019-06-11 Keygene N.V. Method for identifying the source of an amplicon
CN101310024B (en) * 2005-11-14 2012-10-03 科因股份有限公司 Method for high throughput screening of transposon tagging populations and massive parallel sequence identification of insertion sites
EP2789696B1 (en) 2005-12-22 2015-12-16 Keygene N.V. Method for high-throughput AFLP-based polymorphism detection
WO2007087312A2 (en) 2006-01-23 2007-08-02 Population Genetics Technologies Ltd. Molecular counting
EP2963127B1 (en) 2006-04-04 2017-08-16 Keygene N.V. High throughput detection of molecular markers based on restriction fragments
EP2180596A1 (en) 2007-08-06 2010-04-28 Nihon Dempa Kogyo Co., Ltd. Tuning fork-type crystal oscillator and its frequency adjusting method
US8362325B2 (en) 2007-10-03 2013-01-29 Ceres, Inc. Nucleotide sequences and corresponding polypeptides conferring modulated plant characteristics
US20090124758A1 (en) 2007-11-09 2009-05-14 Bridgestone Sports Co., Ltd. Golf ball
US20140051585A1 (en) 2012-08-15 2014-02-20 Natera, Inc. Methods and compositions for reducing genetic library contamination
CA2913236A1 (en) 2013-06-07 2014-12-11 Keygene N.V. Method for targeted sequencing

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0534858A1 (en) 1991-09-24 1993-03-31 Keygene N.V. Selective restriction fragment amplification : a general method for DNA fingerprinting
US6013445A (en) * 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
WO2000024939A1 (en) * 1998-10-27 2000-05-04 Affymetrix, Inc. Complexity management and analysis of genomic dna
WO2000061800A2 (en) * 1999-04-09 2000-10-19 Keygene N.V. Method for the analysis of aflp® reaction mixtures using primer extension techniques
WO2005003375A2 (en) * 2003-01-29 2005-01-13 454 Corporation Methods of amplifying and sequencing nucleic acids
WO2006137733A1 (en) * 2005-06-23 2006-12-28 Keygene N.V. Strategies for high throughput identification and detection of polymorphisms
WO2006137734A1 (en) * 2005-06-23 2006-12-28 Keygene N.V. Improved strategies for sequencing complex genomes using high throughput sequencing technologies

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ALBERT L. LEHNINGER: "Principles of Biochemistry", 1982, WORTH PUB., pages: 793 - 800
BREYNE PETER ET AL: "Transcriptome analysis during cell division in plants.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 99, no. 23, 12 November 2002 (2002-11-12), pages 14825 - 14830, XP002427181, ISSN: 0027-8424 *
LINDSTEDT B A ET AL: "A variation of the amplified-fragment length polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme combination BglII-MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 189, no. 1, 1 August 2000 (2000-08-01), pages 19 - 24, XP002221164, ISSN: 0378-1097 *
MARGULIES MARCEL ET AL: "Genome sequencing in microfabricated high-density picolitre reactors", NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 437, no. 7057, 15 September 2005 (2005-09-15), pages 376 - 380, XP002398505, ISSN: 0028-0836 *
NICOD J-C ET AL: "SNPs by AFLP (SBA): a rapid SNP isolation strategy for non-model organisms", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 31, no. 5, 1 March 2003 (2003-03-01), pages E19-1 - E19-5, XP002398230, ISSN: 0305-1048 *
SHENDURE ET AL., SCIENCE, vol. 309, no. 5741, pages 1728 - 1732
SHENDURE JAY ET AL: "Accurate multiplex polony sequencing of an evolved bacterial genome", SCIENCE (WASHINGTON D C), vol. 309, no. 5741, September 2005 (2005-09-01), pages 1728 - 1732, XP002427180, ISSN: 0036-8075 *

Cited By (119)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009068604A1 (en) * 2007-11-27 2009-06-04 Vib Vzw Marker assisted directed evolution
EP2172565A1 (en) * 2008-09-24 2010-04-07 Cyano Biotech GmbH Method of identifying and/or differentiating cyanophyta
WO2010064897A1 (en) 2008-12-05 2010-06-10 Keygene N.V. Farmesene synthase
US11447822B2 (en) 2009-04-30 2022-09-20 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US11499188B2 (en) 2009-04-30 2022-11-15 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US11499187B2 (en) 2009-04-30 2022-11-15 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US10266884B2 (en) 2009-04-30 2019-04-23 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US11352665B2 (en) 2009-04-30 2022-06-07 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US11339432B2 (en) 2009-04-30 2022-05-24 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US10501793B2 (en) 2009-04-30 2019-12-10 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
US10266883B2 (en) 2009-04-30 2019-04-23 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
WO2011074960A1 (en) * 2009-12-17 2011-06-23 Keygene N.V. Restriction enzyme based whole genome sequencing
US8932812B2 (en) 2009-12-17 2015-01-13 Keygene N.V. Restriction enzyme based whole genome sequencing
US11008607B2 (en) 2010-04-05 2021-05-18 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10662468B2 (en) 2010-04-05 2020-05-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11866770B2 (en) 2010-04-05 2024-01-09 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10494667B2 (en) 2010-04-05 2019-12-03 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11293917B2 (en) 2010-04-05 2022-04-05 Prognosys Biosciences, Inc. Systems for analyzing target biological molecules via sample imaging and delivery of probes to substrate wells
US11001878B1 (en) 2010-04-05 2021-05-11 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11767550B2 (en) 2010-04-05 2023-09-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11761030B2 (en) 2010-04-05 2023-09-19 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11001879B1 (en) 2010-04-05 2021-05-11 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11733238B2 (en) 2010-04-05 2023-08-22 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10308982B2 (en) 2010-04-05 2019-06-04 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11732292B2 (en) 2010-04-05 2023-08-22 Prognosys Biosciences, Inc. Spatially encoded biological assays correlating target nucleic acid to tissue section location
US11634756B2 (en) 2010-04-05 2023-04-25 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11560587B2 (en) 2010-04-05 2023-01-24 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10472669B2 (en) 2010-04-05 2019-11-12 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10480022B2 (en) 2010-04-05 2019-11-19 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11156603B2 (en) 2010-04-05 2021-10-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11549138B2 (en) 2010-04-05 2023-01-10 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11542543B2 (en) 2010-04-05 2023-01-03 Prognosys Biosciences, Inc. System for analyzing targets of a tissue section
US11519022B2 (en) 2010-04-05 2022-12-06 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10612079B2 (en) 2010-04-05 2020-04-07 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10619196B1 (en) 2010-04-05 2020-04-14 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10996219B2 (en) 2010-04-05 2021-05-04 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11067567B2 (en) 2010-04-05 2021-07-20 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10662467B2 (en) 2010-04-05 2020-05-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11313856B2 (en) 2010-04-05 2022-04-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11208684B2 (en) 2010-04-05 2021-12-28 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11479810B1 (en) 2010-04-05 2022-10-25 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10982268B2 (en) 2010-04-05 2021-04-20 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11401545B2 (en) 2010-04-05 2022-08-02 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11384386B2 (en) 2010-04-05 2022-07-12 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11371086B2 (en) 2010-04-05 2022-06-28 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11365442B2 (en) 2010-04-05 2022-06-21 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10914730B2 (en) 2010-04-05 2021-02-09 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10983113B2 (en) 2010-04-05 2021-04-20 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10962532B2 (en) 2010-04-05 2021-03-30 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10961566B2 (en) 2010-04-05 2021-03-30 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20130237432A1 (en) * 2010-06-30 2013-09-12 Jian Li Application of a pcr sequencing method, based on dna barcoding technique and dna incomplete shearing strategy, in hla genotyping
KR20130038353A (en) * 2010-06-30 2013-04-17 비지아이 션전 코포레이션 리미티드 New pcr sequencing method and use thereof in hla genotyping
KR101709826B1 (en) * 2010-06-30 2017-02-24 비지아이 제노믹스 코포레이션 리미티드 New pcr sequencing method and use thereof in hla genotyping
US9957564B2 (en) * 2010-06-30 2018-05-01 Bgi Genomics Co., Ltd. Application of a PCR sequencing method, based on DNA barcoding technique and DNA incomplete shearing strategy, in HLA genotyping
WO2012096579A2 (en) 2011-01-14 2012-07-19 Keygene N.V. Paired end random sequence based genotyping
US11352659B2 (en) 2011-04-13 2022-06-07 Spatial Transcriptomics Ab Methods of detecting analytes
US11479809B2 (en) 2011-04-13 2022-10-25 Spatial Transcriptomics Ab Methods of detecting analytes
US11788122B2 (en) 2011-04-13 2023-10-17 10X Genomics Sweden Ab Methods of detecting analytes
US11795498B2 (en) 2011-04-13 2023-10-24 10X Genomics Sweden Ab Methods of detecting analytes
US12006544B2 (en) 2011-04-15 2024-06-11 The Johns Hopkins University Safe sequencing system
US20240084381A1 (en) * 2011-04-15 2024-03-14 The Johns Hopkins University Safe sequencing system
US20140221217A1 (en) * 2011-07-08 2014-08-07 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US10422001B2 (en) 2011-07-08 2019-09-24 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US10988807B2 (en) 2011-07-08 2021-04-27 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US11873529B2 (en) 2011-07-08 2024-01-16 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US9777322B2 (en) * 2011-07-08 2017-10-03 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US9951384B2 (en) 2012-01-13 2018-04-24 Data2Bio Genotyping by next-generation sequencing
EP2802666A1 (en) 2012-01-13 2014-11-19 Data2Bio Genotyping by next-generation sequencing
US10704091B2 (en) 2012-01-13 2020-07-07 Data2Bio Genotyping by next-generation sequencing
EP2802666B1 (en) * 2012-01-13 2018-09-19 Data2Bio Genotyping by next-generation sequencing
US10450606B2 (en) 2012-02-17 2019-10-22 Fred Hutchinson Cancer Research Center Compositions and methods for accurately identifying mutations
US11441180B2 (en) 2012-02-17 2022-09-13 Fred Hutchinson Cancer Center Compositions and methods for accurately identifying mutations
US11697843B2 (en) 2012-07-09 2023-07-11 Tecan Genomics, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
CN102864498B (en) * 2012-09-24 2014-07-16 中国科学院天津工业生物技术研究所 Establishment method of long mate pair library
CN102864498A (en) * 2012-09-24 2013-01-09 天津工业生物技术研究所 Establishment method of long mate pair library
AU2013326406B2 (en) * 2012-10-05 2019-01-03 Katholieke Universiteit Leuven, KU LEUVEN R&D High-throughput genotyping by sequencing low amounts of genetic material
EP2904113B1 (en) 2012-10-05 2020-02-26 Katholieke Universiteit Leuven K.U. Leuven R&D High-throughput genotyping by sequencing low amounts of genetic material
EP3699292A1 (en) * 2012-10-05 2020-08-26 Katholieke Universiteit Leuven, K.U.Leuven R&D High-throughput genotyping by sequencing low amounts of genetic material
USRE50065E1 (en) 2012-10-17 2024-07-30 10X Genomics Sweden Ab Methods and product for optimising localised or spatial detection of gene expression in a tissue sample
US10619206B2 (en) 2013-03-15 2020-04-14 Tecan Genomics Sequential sequencing
US10760123B2 (en) 2013-03-15 2020-09-01 Nugen Technologies, Inc. Sequential sequencing
US11286515B2 (en) 2013-06-25 2022-03-29 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US10774372B2 (en) 2013-06-25 2020-09-15 Prognosy s Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11359228B2 (en) 2013-06-25 2022-06-14 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11618918B2 (en) 2013-06-25 2023-04-04 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11046996B1 (en) 2013-06-25 2021-06-29 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US10927403B2 (en) 2013-06-25 2021-02-23 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11753674B2 (en) 2013-06-25 2023-09-12 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11821024B2 (en) 2013-06-25 2023-11-21 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11725241B2 (en) 2013-11-13 2023-08-15 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US11098357B2 (en) 2013-11-13 2021-08-24 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
WO2015083004A1 (en) * 2013-12-02 2015-06-11 Population Genetics Technologies Ltd. Method for evaluating minority variants in a sample
US10927408B2 (en) 2013-12-02 2021-02-23 Personal Genome Diagnostics, Inc. Method for evaluating minority variants in a sample
US11390912B2 (en) 2015-04-10 2022-07-19 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US11739372B2 (en) 2015-04-10 2023-08-29 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US11299774B2 (en) 2015-04-10 2022-04-12 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US11162132B2 (en) 2015-04-10 2021-11-02 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US10774374B2 (en) 2015-04-10 2020-09-15 Spatial Transcriptomics AB and Illumina, Inc. Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US11613773B2 (en) 2015-04-10 2023-03-28 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
EP3187040A1 (en) 2015-12-30 2017-07-05 Vilmorin et Cie Resistance to tolcndv in melons
EP3443115B1 (en) * 2016-04-15 2024-10-09 Menarini Silicon Biosystems S.p.A. Method for the generation of dna libraries for massive parallel sequencing
KR20190019049A (en) * 2016-04-15 2019-02-26 메나리니 실리콘 바이오시스템스 에스.피.에이. METHOD AND APPARATUS FOR GENERATING DNA LIBRARIES FOR LARGE PARALLEL SEQUENCES
KR102354422B1 (en) 2016-04-15 2022-01-21 메나리니 실리콘 바이오시스템스 에스.피.에이. Method for generating DNA library for bulk parallel sequencing and kit therefor
US11584958B2 (en) 2017-03-31 2023-02-21 Grail, Llc Library preparation and use thereof for sequencing based error correction and/or variant identification
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
WO2019121603A1 (en) 2017-12-18 2019-06-27 Keygene N.V. Chemical mutagenesis of cassava
WO2020109412A1 (en) 2018-11-28 2020-06-04 Keygene N.V. Targeted enrichment by endonuclease protection
WO2020169830A1 (en) 2019-02-21 2020-08-27 Keygene N.V. Genotyping of polyploids
WO2020243678A1 (en) * 2019-05-31 2020-12-03 North Carolina State University Compositions and methods related to quantitative reduced representation sequencing
WO2021116371A1 (en) 2019-12-12 2021-06-17 Keygene N.V. Semi-solid state nucleic acid manipulation
WO2021123062A1 (en) 2019-12-20 2021-06-24 Keygene N.V. Ngs library preparation using covalently closed nucleic acid molecule ends
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system
WO2022074058A1 (en) 2020-10-06 2022-04-14 Keygene N.V. Targeted sequence addition
WO2022112316A1 (en) 2020-11-24 2022-06-02 Keygene N.V. Targeted enrichment using nanopore selective sequencing
WO2022112394A1 (en) 2020-11-25 2022-06-02 Koninklijke Nederlandse Akademie Van Wetenschappen Ribosomal profiling in single cells
WO2024121354A1 (en) 2022-12-08 2024-06-13 Keygene N.V. Duplex sequencing with covalently closed dna ends
WO2024209000A1 (en) 2023-04-04 2024-10-10 Keygene N.V. Linkers for duplex sequencing

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