WO2012079486A1 - Procédé de préparation d'échantillon d'adn pour séquençage, et son utilisation - Google Patents

Procédé de préparation d'échantillon d'adn pour séquençage, et son utilisation Download PDF

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WO2012079486A1
WO2012079486A1 PCT/CN2011/083726 CN2011083726W WO2012079486A1 WO 2012079486 A1 WO2012079486 A1 WO 2012079486A1 CN 2011083726 W CN2011083726 W CN 2011083726W WO 2012079486 A1 WO2012079486 A1 WO 2012079486A1
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dna
sequencing
fragment
unit
dna fragment
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PCT/CN2011/083726
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English (en)
Chinese (zh)
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吴逵
阿叁
耿春雨
张秀清
杨焕明
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深圳华大基因科技有限公司
深圳华大基因研究院
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Publication of WO2012079486A1 publication Critical patent/WO2012079486A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to the field of biotechnology, and in particular to the field of DNA sequencing technology, and in particular to a method for preparing a DNA sample for sequencing and its use. More specifically, the present invention provides a method of preparing a DNA sample for sequencing, a method of constructing a DNA sequencing library, a DNA sequencing library, a method of DNA sequencing, a method of determining genomic DNA sequence information, and a preparation of a DNA sample for sequencing. Devices and DNA sequencing systems. Background technique
  • the genome of a living being is the sum of the genetic information carried by the organism.
  • the sequencing of the genome can not only study the existence of genes, the structure and function of genes, the relationship between genes, but also the genes such as gene expression and regulation.
  • Basic research provides important data and is of great significance for applied research in disease diagnostics and gene therapy.
  • Shotgun sequencing is currently the commonly used genome sequencing method.
  • the method steps mainly include: fragmenting the genomic DNA DN A to obtain a DNA fragment, and then performing high-throughput sequencing on the DN A fragments to obtain sequence information of the DNA fragment; sequencing with long fragments above lkb, ie Construct a long-length end-paired sequencing library, and then sequence it to obtain sequence information at both ends of the long fragment; use the sequence information of the two ends of the long fragment and the sequence information of the DNA fragment to perform genome assembly to obtain the entire genome The sequence information is ultimately achieved by genome sequencing.
  • the short sequence that is, the contig of the DNA fragment sequence can be effectively assembled into a larger scaffold, which is relatively similar to human or fruit flies.
  • Large and complex genomic assembly is a key breakthrough (see Myers EW, et al: A whole-genome assembly of Drosophila. Science 2000, 287 (5461): 2196-2204., which is incorporated herein by reference in its entirety. ). Therefore, the ability to successfully construct a terminal-paired sequencing library of a large span of fragments is critical to the sequencing of the genome.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides methods of preparing DNA samples for sequencing and uses thereof.
  • the invention provides a method of preparing a DNA sample for sequencing.
  • the method comprises the steps of: fragmenting genomic DNA to obtain a first DNA fragment; performing end repair of the first DNA fragment to obtain a DNA fragment that has been repaired at the end; a fragment is ligated to capture a marker to obtain a DNA fragment having a capture marker; a DNA fragment having a capture marker is cyclized to obtain a circular DNA; a circular DNA is fragmented to obtain a second DNA fragment; and a second The DNA fragment is screened to obtain a fragment of interest which constitutes a DNA sample for sequencing.
  • the target fragment comprises the end sequence of the first DNA fragment.
  • a DNA sample for sequencing of genomic DNA can be conveniently and efficiently prepared by using the method, and the obtained DNA sample can be effectively applied to construct a DNA sequencing library, thereby enabling end pairing of the first DNA fragment.
  • Sequencing library Therefore, based on the high-throughput sequencing technology, the end sequence information of the first DNA fragment can be accurately determined, so that the long fragment of the genomic DNA, that is, the end sequence information of the first DNA fragment can be effectively used for assisting genome assembly, that is, The genome sequence data can be efficiently assembled to obtain complete genomic sequence information.
  • the present invention provides a method of constructing a DNA sequencing library.
  • the method comprises the steps of: preparing a DNA fragment for sequencing according to an embodiment of the present invention, preparing a fragment of interest; performing end repair on the target fragment and adding base A at the 3' end to obtain Adding a target fragment of base A to the end; linking the target fragment to which the base A is added to the linker to obtain a ligation product; PCR-amplifying the ligation product to obtain an amplification product; and isolating and purifying the amplification product, the expansion
  • the amplified product constitutes a DNA sequencing library.
  • a DNA sequencing library constructed by the method for constructing a DNA sequencing library of the present invention can be effectively applied to a high-throughput sequencing platform such as an Illumina sequencing platform, and an DNA sequencing library can be accurately determined based on the sequencing result.
  • the sequence information, based on the sequence information of the DNA sequencing library, can effectively assist in genome assembly.
  • the invention provides a DNA sequencing library.
  • the DNA sequencing library is constructed by a method of constructing a DNA sequencing library according to an embodiment of the present invention. According to this issue
  • the DNA sequencing library can be effectively used in a high-throughput sequencing platform such as an Illumina sequencing platform, and the sequence information of the DNA sequencing library determined based on the sequencing result can effectively assist in genome assembly.
  • the present invention provides a method of DNA sequencing.
  • the method comprises: constructing a DNA sequencing library of genomic DNA according to a method of constructing a DNA sequencing library according to an embodiment of the present invention; and sequencing a DNA sequencing library of genomic DNA to obtain a sequencing result.
  • genomic DNA can be sequenced and reproducible, and the sequencing results are accurate and effective, and can be used for assisting genome assembly, and thus can be applied to subsequent determination of sequence information of genomic DNA.
  • the invention provides a method of determining genomic DNA sequence information.
  • the method comprises the steps of: dividing genomic DNA into a first genomic DNA sample and a second genomic DNA sample; using the first genomic DNA sample, using a method of DNA sequencing according to an embodiment of the present invention A genomic DNA sample is sequenced, and partial sequence information of the genomic DNA is determined based on the sequencing result; the second genomic DNA sample is sequenced according to a conventional sequencing method, and the sequencing data of the genomic DNA is obtained, wherein
  • the conventional sequencing method is at least one selected from the group consisting of SOLEXA, SOLID, 454, and single molecule sequencing techniques; and assembling and splicing partial sequence information of genomic DNA with sequencing data of genomic DNA to determine sequence information of genomic DNA .
  • the inventors have found that the method can effectively determine the sequence information of genomic DNA, and the repeatability is good, and the result is accurate and reliable.
  • the present invention provides an apparatus for preparing a DNA sample for sequencing.
  • the apparatus comprises: a first fragmentation unit for fragmenting genomic DNA to obtain a first DNA fragment; an end repair unit, the end repair unit and the first fragment The unit is ligated for end-repairing the first DNA fragment to obtain a DNA fragment which is end-repaired; a labeling unit, which is connected to the end-repairing unit, for connecting the end-repaired DNA fragment to the capture marker so that Obtaining a DNA fragment having a capture label; a cyclization unit, the cyclization unit being linked to the label unit for cyclizing the DNA fragment having the capture label to obtain a circular DNA; a second fragmentation unit, the second a fragmentation unit coupled to the cyclization unit for fragmenting the circular DNA to obtain a second DNA fragment; and a screening unit coupled to the second fragmentation unit for screening the second DNA fragment,
  • the fragment of interest constitutes a DNA sample for sequencing.
  • the inventors have surprisingly found that with the apparatus for preparing a DNA sample for sequencing according to an embodiment of the present invention, a DNA sample for sequencing can be conveniently and efficiently prepared, and the repeatability is good, and the obtained DNA sample can be effectively applied to DNA.
  • the sequencing library was prepared to be successfully used for high throughput sequencing.
  • the present invention provides a DNA sequencing system.
  • the system comprises: a sample preparation device which is a device for preparing a DNA sample for sequencing according to an embodiment of the present invention, for preparing a fragment of interest, the fragment of interest constituting for sequencing a DNA sample; a library construction device coupled to the sample preparation device for constructing a sequencing library for the DNA sample for sequencing; and a sequencing device coupled to the library construction device for sequencing the sequencing library .
  • the system can effectively sequence genomic DNA samples, and the operation is convenient, rapid, less time-consuming, reproducible, and the sequencing results are accurate and reliable.
  • Figure 1 is a schematic flow diagram showing a method of constructing a DNA sequencing library according to one embodiment of the present invention.
  • Fig. 2 is a view showing an electrophoretogram of a DNA fragment obtained by disrupting penguin genomic DNA using a standard Hydroshear instrument with different interruption parameters according to Example 1 of the present invention.
  • Fig. 3 is a chart showing the electrophoresis of a 40 - 45 kb biotin-labeled DNA fragment obtained by electrophoresis separation in Example 1 of the present invention.
  • Figure 4 shows the result of the insertion range verification on the penguin genome aligned to the paired end sequence obtained in Example 1 of the present invention.
  • Figure 5 shows the result of the insertion range verification on the paired-end sequence obtained in Example 2 according to the present invention.
  • Fig. 6 shows the result of the insertion range verification on the paired end sequence obtained in Example 3 according to the present invention.
  • Figure 7 A schematic diagram showing an apparatus for preparing a DNA sample for sequencing according to one embodiment of the present invention.
  • Figure 8 shows a DNA sequencing system of one embodiment of the present invention.
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first”, “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, “multiple” means two or more unless otherwise stated.
  • the invention provides a method of preparing a DNA sample for sequencing. According to an embodiment of the invention, the method comprises the steps of:
  • the genomic DNA is fragmented to obtain a first DNA fragment.
  • the step of extracting genomic DNA from the sample may be further included.
  • the source of the sample is not particularly limited.
  • the sample may be derived from at least one of an animal, a plant, and a living organism.
  • the animal may be at least one of a human and a penguin.
  • the plant may be plum.
  • the method of fragmenting genomic DNA is not particularly limited.
  • fragmentation of genomic DNA can be carried out by at least one selected from the group consisting of a misting method, a sonication method, a HydroShear, and a digestion treatment.
  • genomic DNA can be fragmented using a HydroShear instrument.
  • the method further comprises: performing segment selection on the first DNA fragment.
  • the length of the first DNA fragment is not particularly limited, and preferably, the length of the first DNA fragment is 20-50 kb, and more preferably, the length of the first DNA fragment is 25-50 kb.
  • the first DNA fragment is end-repaired to obtain a DNA fragment that has been repaired at the end.
  • the first DNA fragment can be end-repaired using Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase, wherein the Klenow fragment has 5 ' ⁇ 3 'polymerase activity and 3 ' ⁇ 5 ' Polymerase activity, but lacks 5' ⁇ 3' exonuclease activity.
  • capture marker refers to a marker by which a DNA fragment can be labeled, by virtue of the nature of the marker, for example, by means of an agent that specifically binds to the marker.
  • the captured DNA fragments are screened and screened. According to an embodiment of the present invention, the type of the capture mark is not particularly limited.
  • the capture label for use is biotin, whereby by using biotin-labeled DNA fragments, reagents that can specifically bind biotin can be used in subsequent operations, such as carrying streptavidin
  • the magnetic beads which can specifically bind to biotin
  • the inventors have found that the introduction of biotin into the DNA fragment does not affect subsequent processing, thereby improving the efficiency of sequencing library construction, thereby improving the efficiency and accuracy of sequencing.
  • a capture marker can be ligated using a Klenow fragment, T4 DNA polymerase, and a T4 polynucleotide kinase, wherein the Klenow fragment has 5' ⁇ 3' polymerase activity and 3' ⁇ 5' polymerase activity, but Lack of 5' ⁇ 3 'exonuclease activity.
  • it may further comprise: performing fragment selection on the DNA fragment having the capture marker.
  • the method of segment selection is not particularly limited. Root According to some specific examples of the invention, fragment selection can be performed using 0.6% agarose electrophoresis.
  • the DNA fragment having the capture marker may be 20-50 kb in length.
  • the DNA fragment having the capture marker is subjected to a cyclization treatment to obtain a circular DNA.
  • a DNA fragment having a marker can be subjected to a cyclization treatment using T4 DNA ligase and T3 DNA ligase.
  • the step of removing the uncircularized DNA fragment may be further included.
  • the method of removing the uncircularized DNA fragment is not particularly limited.
  • the uncircularized DNA fragment can be removed by using at least one selected from the group consisting of a DNase and an exonuclease.
  • the DNase is an ATP-dependent DNase which does not degrade the plasmid
  • the exonuclease is exonuclease I.
  • the circular DNA is fragmented to obtain a second DNA fragment.
  • the ring will be
  • the method of DNA fragmentation is not particularly limited. According to some specific examples of the present invention, fragmentation of the circular DNA can be carried out by at least one selected from the group consisting of an atomization method, a sonication method, a HydroShear, and a digestion treatment. According to one embodiment of the invention, the circular DNA can be fragmented using a Covaris ultrasonic interrupter. According to an embodiment of the invention, the second DNA fragment may be from 100 to 1000 bp in length. According to some specific examples of the invention, the second DNA fragment may be 200-800 bp.
  • the second DNA fragment is screened to obtain a fragment of interest which constitutes a DNA sample for sequencing.
  • the method of screening the second DNA fragment is not particularly limited.
  • screening the second DNA fragment is performed using magnetic bead capture, wherein the magnetic bead carries a molecular entity capable of specifically recognizing the captured label.
  • the capture marker is a biotin and the molecular entity carried on the magnetic bead is streptavidin.
  • a DNA sample for sequencing of genomic DNA can be conveniently and efficiently prepared by using the method, and the obtained DNA sample can be effectively applied to construct a DNA sequencing library, thereby obtaining a DNA sequencing library of genomic DNA (also It may be referred to as a terminal paired sequencing library, wherein "end” refers herein to both ends of the first DNA fragment).
  • a DNA sequencing library of genomic DNA also It may be referred to as a terminal paired sequencing library, wherein "end” refers herein to both ends of the first DNA fragment.
  • the present invention provides a method of constructing a DNA sequencing library. According to an embodiment of the invention, referring to Figure 1, the method comprises the following steps:
  • a method of preparing a DNA sample for sequencing according to an embodiment of the present invention a target fragment is prepared.
  • the target fragment was subjected to terminal repair and base A was added to the 3' end to obtain a target fragment in which the base A was added at the end.
  • the method of performing end repair of the target fragment is not particularly limited.
  • the target fragment can be end-repaired using Klenow fragment, T4 DNA polymerase, and T4 polynucleotide kinase, wherein the Klenow fragment has 5' ⁇ 3' polymerase activity and 3' ⁇ 5' polymerase activity. , but lacks 5 ' ⁇ 3' exonuclease activity.
  • the method of adding the base A to the 3' end of the target fragment is not particularly limited.
  • Klenow (3 '-5' exo-) can be used to carry out the 3' end of the target fragment to base A.
  • the target fragment to which the base A is added at the end is attached to a linker to obtain a ligation product.
  • the method of attaching the target fragment to which the terminal A is added to the terminal is not particularly limited.
  • the target fragment to which the terminal A is added at the end is linked to a linker by using T4 DNA ligase.
  • the ligation product is subjected to PCR amplification to obtain an amplification product.
  • the amplified product is isolated and purified, and the amplified product constitutes a DNA sequencing library.
  • the method of separating and purifying the amplification product is not particularly limited.
  • the amplified product can be isolated and purified by 2% agarose gel electrophoresis.
  • a DNA sequencing library can be constructed simply, quickly, and efficiently, and the reproducibility is good, and the resulting library is of very good quality.
  • the inventors have surprisingly found that DNA sequencing libraries constructed by this method can be effectively applied to high-throughput sequencing platforms such as the Illumina sequencing platform, and based on the sequencing results, the sequence information of the DNA sequencing library can be accurately determined, based on the DNA sequencing library. The sequence information can effectively assist in genome assembly.
  • the method of constructing a DNA sequencing library of the present invention may comprise the following steps:
  • it may also include
  • step 6 the step of adding the base A and the ligation-splicing link to the DNA fragment after the end-filling in step 6), In order to obtain a ligation product;
  • it may also include
  • the genomic DNA in step 1), can be interrupted into a 25 - 50 kb DNA fragment.
  • genomic DNA can be interrupted into a 20 - 40 kb DNA fragment, a 30 - 50 kb DNA fragment, a 35 - 50 kb DNA fragment, a 40 - 50 kb DNA fragment, or a 40 - 45 kb DNA fragment.
  • the sample genomic DNA may be genomic DNA of any species including, but not limited to, mammals, birds, or plants (eg, dicots), specifically including primates, penguins, or rosettes. More specifically, including human, penguin, or Rosaceae (such as Prunus).
  • the sample genomic DNA can be human, penguin (eg, Pygoscelis adeliae), or plum (eg, wild) Plum, (Prunus mume) genomic DNA.
  • genomic DNA can be physically interrupted, for example, by atomization, ultrasonic fragmentation, or interruption using a HydroShear instrument to break genomic DNA into fragments of 20 _ 50 kb in size.
  • the HydroShear apparatus is used for interrupting, and by adjusting the speed of the flow through the contraction hole and the pore size of the contraction hole, the size of the fragment after the genomic DNA is interrupted can be controlled, and the genomic DNA is interrupted into a relatively uniform fragment.
  • the HydroShear instrument can be used for interruption, wherein a large segment interrupting accessory can be used, the speed parameter is set to 14 - 16 , and the number of cycles is set to 30 - 40 (different values are selected according to the segment size),
  • the range of disrupted fragments of genomic DNA can be increased to 20 _ 50 kb.
  • the separation in step 2), may be gel electrophoresis separation; specifically, it may be separated by agarose gel electrophoresis, and may be performed by ordinary agarose gel electrophoresis or pulsed-field gel electrophoresis. Then, using a gelatin recovery, the DNA fragment of the desired size is isolated and purified.
  • the capture label can be a biotin, and correspondingly, the separation in step 5) can be carried out by using magnetic beads with streptavidin.
  • step 2) and step 5) may also be carried out based on a binding system similar to the antibody-antigen reaction.
  • a polymerase such as Klenow large fragment enzyme, T4 DNA polymerase and T4 polynucleotide kinase and dNTPs fill the ends to produce blunt-ended DNA.
  • T4 DNA polymerase can smooth the 3' overhanging end, fill the 5' end, and Klenow large fragment enzyme can fill the 5' overhang or excise the 3' overhang, while T4 polynucleotide kinase will The 5' end is phosphorylated and the 3' terminal phosphate group is removed for the ligation reaction.
  • step 2) after finishing the end of the DNA fragment, the end-filled DNA fragment can be labeled with Biotin, wherein the labeled reaction system and conditions are filled with the end.
  • the reaction was similar, except that the common dNTP was replaced with a mixture of Biotin-dNTP and common dNTP, and then the 3'-5' exonuclease activity and 5 '-3 'polymerase activity of Klenow large fragment enzyme, T4 DNA polymerase were utilized. , a substitution reaction occurs at the 3' end of the DNA fragment, The common dNTP was replaced with Biotin-dNTP, thereby labeling the biotin with a DNA fragment that was maintained at the blunt end.
  • the isolated DNA fragment of the desired size is cyclized, and the two ends of the DNA of the target fragment can be formed by the combination of T4 DNA ligase and T3 DNA ligase. Connect to make the segment loop.
  • ligation can also be carried out using T4 DNA ligase or T3 DNA ligase alone, according to an embodiment of the present invention, in a PEG-containing ligation buffer, incubated at 16 ° C for 16 hours.
  • T3 DNA ligase and T4 DNA ligase were used in combination.
  • the effect of cyclization efficiency (referring to the ratio of fragmented linear DNA self-ligated into circular DNA) from 1% - 3% to 5% - 10% can be achieved by using T3 DNA ligase alone or T4 DNA ligase alone. Therefore, it is preferred to carry out the ligation and cyclization treatment using a combination of T3 DNA ligase and T4 DNA ligase.
  • a step of performing a water bath immediately after the incubation of the DNA mixture at 50 - 75 ° C for 1 to 30 minutes is carried out before the cyclization reaction. This step reduces the chances of linking different DNA fragments together, ensuring that each cyclized DNA molecule is a single fragment.
  • the incubation temperature may be 60-70 ° C, such as 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 ° C
  • the incubation time may be 5 - 25 minutes, more specifically, the incubation time may be 10 - 20 minutes, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.
  • the DNA mixture was incubated at 65 ° C for 15 minutes and immediately after a water bath.
  • the unligated fragmented DNA needs to be removed, otherwise the sequencing of the paired-end library is affected.
  • the unligated fragmented DNA can be removed by a known method of digesting linear DNA.
  • an unconjugated double-stranded or single-stranded DNA can be degraded using a plasmid-safe ATP-dependent DNase or an exonuclease I.
  • high-efficiency self-ligation cyclization of the blunt end of the DNA fragment can be utilized, whereby the steps of designing the cleavage site required for the use of the foreign vector or introducing the intermediate link to realize the cyclization connection can be omitted.
  • Randomly interrupting the process of fragmenting circular DNA can greatly increase the availability of sequencing data at the paired ends. This is because if the enzymatic cleavage method is used to interrupt the paired end sequence obtained as described above, the read length is too short (only about 25 bp is effective at each end).
  • the efficient self-ligation cyclization of the DNA fragment is used, and the two ends of the connection point are the genomic sequence information, and there are no other external sequences or intermediate connectors, so that the data information can be utilized to the utmost extent (the effective data at each end can reach lOObp) or above).
  • circular DNA can be fragmented using various known breaking methods, and according to a specific example of the present invention, circular DNA can be fragmented by atomization, sonication or HydroShear. According to one embodiment of the present invention, it is preferred to use a Covaris S2 instrument for ultrasonic disruption to break a 20-40 kb circular DNA into a linear DNA fragment of 200 - 800b. Not all of the linear DNA fragments obtained by these interruptions are paired end fragments required for sequencing.
  • the ligation capture marker (biotin tag) performed in step 2) is a substitution tag for several bases at the end of the fragment, so only the end of the fragment carries biotin, and after the cyclization, these biotin-labeled ends are Linked together, these biotin-labeled paired-end fragments can be specifically captured by streptavidin magnetic beads, while those without biotin-labeled intermediate fragments are unable to It is removed in combination with the magnetic beads.
  • the circular DNA in step 4), can be interrupted into a DNA fragment of 100 - l,000b, and according to an embodiment of the present invention, a fragment of 200 _ 800 bp can be interrupted, according to the present invention.
  • the segment of 200-700b can be interrupted.
  • the segment of 200-600 bp can be interrupted; according to another embodiment of the present invention, it can be interrupted to 200. _ 500bp fragment.
  • step 6) _ 8 the DNA fragment captured on the magnetic beads needs to be end-filled, and according to an embodiment of the present invention, a polymerase such as Klenow large fragment enzyme, T4 DNA polymerase and T4 can also be used. Nucleotide kinases and dNTPs fill the ends to produce blunt-ended DNA, which can then be added to the 3' end of the DNA fragment using Klenow (3'-5'exo-) polymerase and dATP, where Klenow ( 3'-5'exo-) polymerase retains DNA polymerase activity but loses 5'-3' and 3'-5' exonuclease activity.
  • Klenow large fragment enzyme T4 DNA polymerase and T4
  • Klenow ( 3'-5'exo-) polymerase retains DNA polymerase activity but loses 5'-3' and 3'-5' exonuclease activity.
  • the sequencing linker can be ligated to the end of the DNA fragment by using T4 DNA ligase, and the T base swell at the end of the linker and the A base highlight complementary pair at the end of the DNA fragment are used for the ligation.
  • the linker can be selected from Illumina, SOLiD or 454 sequencing adaptors to accommodate sequencing sequencing of different sequencing platforms.
  • the paired end fragments can be enriched by specific primers after ligation of the linker, which form a sequencing library.
  • the obtained sequencing library can be unidirectionally or bidirectionally sequenced on a second generation sequencing platform such as Illumina, SOLiD or 454 to obtain sequence information of two paired ends and then used for genomic mapping. Assemble or compare.
  • a second generation sequencing platform such as Illumina, SOLiD or 454 to obtain sequence information of two paired ends and then used for genomic mapping. Assemble or compare.
  • the invention provides a DNA sequencing library.
  • the DNA sequencing library was constructed by a method of constructing a DNA sequencing library according to an embodiment of the present invention.
  • the inventors have found that the DNA sequencing library is of high quality and high purity, can be effectively used in high-throughput sequencing platforms such as the Illumina sequencing platform, and the sequence information of the DNA sequencing library determined based on the sequencing results can effectively assist the genome of the source. Assembly.
  • the present invention provides a method of DNA sequencing. According to an embodiment of the invention, the method comprises:
  • a DNA sequencing library of genomic DNA is constructed by the method of constructing a DNA sequencing library according to an embodiment of the present invention.
  • a method of sequencing a DNA sequencing library of genomic DNA is not particularly limited.
  • sequencing can be performed using a high throughput sequencing platform (also referred to as "high throughput sequencing technology").
  • the sequencing may be performed using at least one selected from the group consisting of a second generation sequencing platform and a single molecule sequencing platform.
  • the second generation sequencing platform may be at least one selected from the group consisting of an Illumina-Solexa sequencing platform, an ABI-Solid sequencing platform, and a Roche-454 sequencing platform
  • the single molecule sequencing platform may be selected from the group of Helicos.
  • Real single-molecule sequencing platform Pacific Biosciences' single-molecule real-time sequencing platform and at least one of Oxford Nanopore Technologies' nanopore sequencing platforms.
  • the method may further comprise: determining partial sequence information of the genomic DNA based on the sequencing result.
  • genomic DNA can be efficiently sequenced, and the repeatability is good, the sequencing result is accurate and reliable, and the sequencing result can be used for assisting genome assembly, thereby enabling Applied to subsequent sequence information for determining genomic DNA.
  • the invention provides a method of determining genomic DNA sequence information. According to an embodiment of the invention, the method comprises the steps of:
  • the genomic DNA is divided into a first genomic DNA sample and a second genomic DNA sample.
  • the first genomic DNA sample is sequenced by the method of DNA sequencing according to an embodiment of the present invention, and based on the sequencing result, partial sequence information of the genomic DNA is determined.
  • the second genomic DNA sample is sequenced according to a conventional sequencing method, and the sequencing data of the genomic DNA is obtained, wherein the conventional sequencing method is selected from the group consisting of SOLEXA, SOLID, 454, and single molecule sequencing. At least one of the technologies.
  • the partial sequence information of the genomic DNA and the sequencing data of the genomic DNA are assembled and spliced to determine the sequence information of the genomic DNA.
  • the inventors have found that the method for determining genomic DNA sequence information according to an embodiment of the present invention can effectively determine the sequence information of genomic DNA, and has good repeatability, high accuracy and high reliability.
  • Apparatus and DNA sequencing system for preparing DNA samples for sequencing are provided.
  • an apparatus 1000 for preparing a DNA sample for sequencing includes: a first fragmentation unit 100, an end repair unit 200, a labeling unit 300, a cyclization unit 400, and a second fragmentation unit 500. And a thinning unit 600.
  • the first fragmentation unit 100 is for fragmenting genomic DNA to obtain a first DNA fragment.
  • any device suitable for fragmenting genomic DNA can be used as the first fragmentation unit 100.
  • a HydroShear instrument can be employed as the first fragmentation unit 100.
  • the end repair unit 200 is coupled to the first fragmentation unit 100 for end-repairing the first DNA fragment to obtain an end-repaired DNA fragment.
  • the labeling unit 300 is coupled to the end repair unit 200 for attaching the end-repaired DNA fragment to the capture label to obtain a DNA fragment having the capture label.
  • biotin is provided in the marking unit 300.
  • the cyclization unit 400 is coupled to the labeling unit 300 for cyclizing the DNA fragment having the capture label to obtain circular DNA.
  • the second fragmentation unit 500 is coupled to the cyclization unit 400 for fragmenting the circular DNA to obtain a second DNA fragment, wherein the second fragmentation unit 500 can be interrupted by Covaris ultrasound according to an embodiment of the present invention. instrument.
  • the screening unit 600 is coupled to the second fragmentation unit 500 for screening the second DNA fragment to obtain a fragment of interest which constitutes a DNA sample for sequencing.
  • the screening unit 600 is provided with a magnetic bead carrying streptavidin.
  • the apparatus may further comprise: a genome extraction unit coupled to the first fragmentation unit 100 for extracting genomic DNA from the biological sample.
  • the inventors have surprisingly found that with the apparatus for preparing a DNA sample for sequencing according to an embodiment of the present invention, a DNA sample for sequencing can be conveniently and efficiently prepared, and the reproducibility is good, and the obtained DNA sample is of good quality and high purity. It can be effectively applied to the preparation of DNA sequencing libraries, which can be successfully used for high-throughput sequencing.
  • a DNA sequencing system 10000 includes: a sample preparation device 1000, a library construction device 2000, and a sequencing device 3000.
  • the sample preparation device 1000 is a device for preparing a DNA sample for sequencing according to an embodiment of the present invention, for preparing a fragment of interest, which constitutes a DNA sample for sequencing.
  • Library construction device 2000 is coupled to sample preparation device 1000 for constructing a sequencing library for DNA samples for sequencing.
  • the library construction apparatus 2000 may further include: an end modification unit for connecting the target fragment to the linker to obtain a ligation product; and a PCR amplification unit connected to the end modification unit for The ligation product is amplified to obtain an amplification product; and a purification unit is coupled to the PCR amplification unit for isolating and purifying the amplification product, and the amplification product constitutes a DNA sequencing library.
  • the sequencing device 3000 is coupled to the library construction device 2000 for sequencing the sequencing library. According to an embodiment of the present invention, any device suitable for sequencing a sequencing library can be used as a sequencing Device 3000.
  • the sequencing device 3000 may be at least one selected from the group consisting of a second generation sequencing platform and a single molecule sequencing platform.
  • the second generation sequencing platform may be at least one selected from the group consisting of an Illumina-Solexa sequencing platform, an ABI-Solid sequencing platform, and a Roche-454 sequencing platform
  • the single molecule sequencing platform may be selected from Helicos.
  • the genomic DNA sample can be efficiently sequenced by using the system, and the operation is convenient, rapid, less time-consuming, reproducible, and the sequencing result is accurate and reliable.
  • Example 1 DNA sequencing library construction and sequencing of penguin genome
  • a DNA sequencing library was constructed using 50 ⁇ of genomic DNA of Pygoscelis adeliae as a library sample.
  • the DNA sequencing library is a 40-45 kb insert end-pair library.
  • the genomic DNA was configured to interrupt the reaction system, and then different breaking parameters (speed parameters and number of cycles) were set using the standard Hydroshear instrument (GeneMachine, San Carlos, CA., USA) as the individual treatments of the experiment, both of which were The reaction system is interrupted to interrupt the genomic DNA to obtain a DNA fragment.
  • the DNA fragments obtained by the above experimental treatments were subjected to electrophoresis to examine the breaking effect of each experimental treatment on genomic DNA, and the electrophoresis results are shown in Fig. 2.
  • the schematics of each lane and the sample loading were: Lane 1, molecular weight standard ⁇ -Hind III digest (Takara, Cat. No.
  • Lane 2 original genomic DNA, loading 150 ng; Lane 3, The molecular weight standard Low Range PFG Marker (NEB, item number M0350S); Lane 4, the speed parameter is 14, the number of cycles is 40, the loading is 200ng; the lane 5, the speed parameter is 14, the number of cycles is 30 Interruption effect, loading volume 200ng; Lane 6, molecular weight standard lkb DNA Extension Ladder (Invitrogen, Cat. No.
  • the experimental treatment shown in lane 8 has the best interruption effect, that is, the interruption parameter with the speed parameter of 15 and the number of cycles of 30, and the interruption of the genomic DNA by the standard Hydroshear instrument is the most interrupting effect.
  • the interruption parameter with the speed parameter of 15 and the number of cycles of 30 is the most interrupting effect.
  • the interruption of the genomic DNA by the standard Hydroshear instrument is the most interrupting effect.
  • the breaking parameter of the standard Hydroshear instrument was set to a speed of 15 and a cycle number of 30, and then the ⁇ interrupting reaction system was interrupted to obtain a 20-50 kb DNA fragment.
  • the DNA fragment was recovered and placed in an EP tube, and then the DNA fragment was purified using Agencourt AM purified magnetic beads (Agencourt AMPure Beads, BECKMAN COULTER). Specifically, 1.8 volumes of Agencourt AM purified magnetic beads were added to the EP tube, upside down.
  • the supernatant was transferred to a new manifold, and the magnetic beads were resuspended in the original tube by adding 185 ⁇ l elution buffer.
  • the original tube was also placed at room temperature for 10 minutes, and then transferred to a magnetic stand. Set for 2 minutes, then transfer the supernatant to a new tube, the purpose of which is to maximize the recovery of DNA fragments bound to the beads.
  • the supernatants were combined to obtain a purified DNA fragment, which was prepared for use.
  • Biotin-dNTP 25 ⁇ 1 ⁇ 4 DNA polymerase (3000 units/ml, Enzymatics, Beverly, MA., USA), 5 ⁇ 1 Klenow polymerase (5000 units/ml, Enzymatics) and 25 ⁇ 1 ⁇ 4 polynucleotide kinase (10000 units/ Ml, Enzymatics), incubation at 20 °C for 30 minutes to obtain the biotinylated product, ready for use.
  • the 40-45 kb biotin-labeled DNA fragment obtained by electrophoresis described above was subjected to electrophoresis, and the results are shown in Fig. 3.
  • the schematics of each lane and the sample loading are: Lane 1, molecular weight standard lkb DNA Extension Ladder (Invitrogen, Cat. No. 1051 1-012); Lane 2, 40 - 45 kb organisms obtained by electrophoresis separation The labeled DNA fragment was loaded with about 50 ⁇ ; Lane 3, molecular weight standard lkb DNA Extension Ladder (Invitrogen, Cat. No. 10511-012); Lane 4, molecular weight standard Low Range PFG Marker (NEB, Cat. No. M0350S). As can be seen from Fig. 3, the 40-45 kb biotin-labeled DNA fragment obtained by electrophoresis was qualified.
  • the 40 _ 45 kb biotin-labeled DNA fragment obtained in the previous step was cyclized according to the following procedure: 2000 ⁇ l 2 ⁇ ligase buffer, ⁇ ⁇ 4 DNA ligase was added to a solution of 1000 ng 40 - 45 kb biotin-labeled DNA fragment ( 400,000 units/ml, NEB), ⁇ ⁇ 3 DNA ligase (300,000 units/ml, Enzymatics), then fill the reaction system to 4ml with ultrapure water, and dispense into 8 1.5ml EP tubes, each The tube was 500 ⁇ l so that the DNA concentration in the reaction system was 0.25 ng/l, and then the EP tube was incubated at 16 ° C for 18 hours.
  • each EP tube was placed at 37 °C for 30 minutes to digest the double-stranded or single-stranded linear DNA without cyclization, and then At 75.
  • the enzyme was inactivated by placing it for 20 minutes under C, and 16 ⁇ l of 0.5 ⁇ EDTA was added to inhibit the enzyme activity, and then it was subjected to a water bath for 3 minutes to renature the DNA to obtain a circular DNA.
  • the circular DNA was broken into 200-800b linear DNA fragments using Covaris, and then purified by QIAGEN Mini elution PCR purification kit and dissolved in 50 ⁇ 1 elution buffer to obtain purified DNA fragments. , spare.
  • the purified DNA fragments were mixed with the resuspended magnetic beads in a medium volume in a centrifuge tube, and then incubated on a Thermomixer for 15 minutes at 20 ° C (15 s, 2 rpm, 500 rpm). At this time, the biotin-labeled paired-end fragment in the purified DNA fragment was specifically bound to the magnetic beads, and the biotin-labeled DNA fragment could not be bound to the magnetic beads. Then, the magnetic beads were purified by magnetic bead washing buffer I and elution buffer on the magnetic separation rack as follows: The centrifuge tube was allowed to stand on the magnetic separation rack for 1 minute, and the supernatant was discarded, using 200 ⁇ M.
  • Magnetic beads washing buffer I wash the magnetic beads, re-suspend the magnetic beads five times with each wash, remove the supernatant, and then repeatedly wash the magnetic beads twice with magnetic beads washing buffer I, then centrifuge the tubes Allow to stand on the magnetic separator for 1 minute, discard the supernatant, wash the beads twice with 200 ⁇ of elution buffer, gently resuspend the beads five times with each wash, and then remove the elution of the last wash. To flush, resuspend the magnetic beads by adding 50 ⁇ M of elution buffer to the centrifuge tube to obtain a resuspended magnetic bead DNA solution for use.
  • PCR amplification (a) 98 ° C for 30 seconds; (b) 98 ° C for 10 seconds; (c) 65 ° C for 30 seconds; (d) 72 ° C for 40 seconds; wherein steps (b) to (d) Eighteen cycles, (e) 72 °C for 5 minutes, in order to obtain an amplification product that constitutes a DNA sequencing library of the penguin genome, ie, a terminal paired library of the 40-45 kb insert of the penguin genome.
  • the PCR tube was then stored at 4 ° C and used. 2. Sequencing on the machine
  • the PCR tube was allowed to stand on a magnetic separation rack for 1 minute, and the supernatant was taken out and transferred to a new 1.5 ml EP tube, and the amplified product was placed in the EP tube, and then 2.0% Low Range Ultra agarose gel was used.
  • the amplified product was subjected to electrophoresis, specifically, electrophoresis was carried out for 16 hours at a pulsed field of a voltage of 15 V/cm, and then the gel was stained with ethidium bromide (EB), and a DNA fragment of 400 bp - 700b in length was cut out under a Darkreader.
  • the Qiagen MinElute Gel Purification Kit was then used for gel recovery and purification to obtain a DNA sequencing library of the penguin genome for use.
  • the above DNA sequencing library was sequenced using an Illumina HiSeq 2000 sequencing platform with a sequence of 50 cycles.
  • the insertion fragment of the penguin genome was obtained with 40 kb of paired-end sequence information, using SOAPdenovo software (for example, htt://soa.genomics.org.cn/soapdenovo). Html download), these data were compared to the genomic sequence of the crane, and the distance span of the paired end sequences obtained by sequencing the library on the genome was verified.
  • Fig. 4 shows the results of the insertion range verification of the paired end sequences obtained in this example on the penguin genome.
  • the paired end sequence obtained in this embodiment has a distance span of 40 kb, which is in line with the expected range of the fragment.
  • scaffold N50 In the mapping process (or assembly process) of the genome map, scaffold N50 is an important indicator for evaluating the level of assembly. Genomic assembly firstly splicing DNA fragment sequences into overlapping groups of longer sequences by overlapping relationships. These contigs are contig, and then some information can be determined by cleavage site information or other "marker" information that can determine the alignment or order relationship.
  • the contig is spliced to form a linear arrangement or a relative positional relationship of each contig on the chromosome, that is, to form a scaffokL N50, that is, a contig length of a maximum sequence covering 50% of all nucleotides, sorting contig or scaffold from large to small. And accumulate its length. When the accumulated length reaches half of the total contig or scaffold length, the length of the last contig or scaffold is contig N50 or scaffold N50.
  • a scaffokL N50 that is, a contig length of a maximum sequence covering 50% of all nucleotides, sorting contig or scaffold from large to small. And accumulate its length. When the accumulated length reaches half of the total contig or scaffold length, the length of the last contig or scaffold is contig N50 or scaffold N50.
  • the DNA sequencing library of the Pmnus mume genome was constructed and sequenced in the same manner as in Example 1 to obtain a DNA sequencing library of the plum genome (the end of a 40 kb long fragment) Sequencing results of the paired DNA library).
  • Fig. 5 shows the result of the insertion range verification of the paired terminal sequence obtained in the present example on the plum genome.
  • the distance of the paired terminal sequence obtained in this embodiment is 40 kb, which is in line with the expected range of the segment.
  • the plum genome assembly was performed using SOAPdenovo software.
  • the insert of the plum genome obtained above was used to assemble the 40 kb terminal sequence information.
  • the assembly result was as follows: sc affold N50 was significantly increased to 970 kb.
  • Example 3 DNA library construction and sequencing of the human genome
  • the DNA sequencing library of the human genome was constructed and sequenced in the same manner as in Example 1 to obtain a sequencing sequence of a human genome DNA sequencing library (a 40 kb long fragment end-pair DNA library). result.
  • Fig. 6 shows the results of the insertion range verification of the paired end sequences obtained in the present example aligned to the human genome.
  • the distance of the paired end sequence obtained in this embodiment spans 40 kb, which is in line with the expected range of the fragment.
  • Method for preparing DNA sample for sequencing can be effectively applied to the construction and sequencing of end-paired sequencing libraries of long fragments, and The obtained library was of good quality and the sequencing results were accurate.
  • the end-sequencing of large-span sequences on the genome is realized by constructing a terminal paired library, and the whole experimental process is simple and rapid, and the construction period of one library is only 3 days, and the use of fosmid clone end sequencing has a significant time advantage. , avoiding cumbersome experimental steps and reducing the risk of library construction failure.
  • the valid data obtained for assembly can effectively increase the length of scaffold N50, and promote the level of genome assembly to reach the standard of fine map or even complete map.

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Abstract

La présente invention concerne un procédé de préparation d'un échantillon d'ADN pour séquençage, un procédé de construction d'une bibliothèque de séquençage d'ADN, une bibliothèque de séquençage d'ADN, un procédé de séquençage d'ADN, un procédé de détermination d'informations de séquence d'ADN génomique, un dispositif de préparation d'un échantillon d'ADN pour séquençage, et un système de séquençage d'ADN. Ledit procédé de préparation d'un échantillon d'ADN pour séquençage comprend les étapes suivantes : fragmentation de l'ADN génomique et obtention de premiers fragments d'ADN ; réparation de l'extrémité des premiers fragments d'ADN et obtention de fragments d'ADN dont l'extrémité est réparée ; liaison de marqueurs de capture aux fragments d'ADN dont l'extrémité est réparée et obtention de fragments d'ADN porteurs des marqueurs de capture ; circularisation des fragments d'ADN qui portent les marqueurs de capture et obtention d'ADN circulaire ; fragmentation de l'ADN circulaire et obtention de seconds fragments d'ADN ; et criblage des seconds fragments d'ADN pour obtenir des fragments cibles, lesdits fragments cibles formant l'échantillon d'ADN pour le séquençage.
PCT/CN2011/083726 2010-12-16 2011-12-08 Procédé de préparation d'échantillon d'adn pour séquençage, et son utilisation WO2012079486A1 (fr)

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