WO2012079486A1 - Method of preparing dna sample for sequencing and use thereof - Google Patents

Abstract

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 the sequence information of genomic DNA, a device for preparing a DNA sample for sequencing and a DNA sequencing system are provided. Wherein, the method of preparing a DNA sample for sequencing includes the following steps: fragmenting the genomic DNA and obtaining the first DNA fragments; end-repairing the first DNA fragments and obtaining the end-repaired DNA fragments; linking the capturing labels to the end-repaired DNA fragments and obtaining the DNA fragments carrying the capturing labels; Circularizing the DNA fragments which carry the capturing labels and obtaining the circular DNA; Fragmenting the circular DNA and obtaining the second DNA fragments; screening the second DNA fragments in order to obtain the target fragments, and the target fragments form the DNA sample for sequencing.

Description

 Method for preparing DNA sample for sequencing and application thereof

Priority information

 Priority is claimed on Japanese Patent Application No. 2010-10591448.7, filed on Dec. Technical field

 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. At this stage, 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. Among them, by using the sequence information of the two ends of the long segment, 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.

However, the current method of constructing end-paired sequencing libraries of long fragments (sometimes referred to as "fragments with larger spans") remains to be improved. Summary of the invention

 The present invention has been completed based on the following findings of the inventors:

 At this stage, the method of constructing long-end paired paired sequencing libraries is time-consuming and costly, and it is difficult to prepare end-paired sequencing libraries of fragments up to 20 kb or even 50 kb in length, and sequencing data processing is difficult, which is not conducive to large-scale libraries. Preparation and sequencing.

 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.

 According to one aspect of the invention, 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: 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. Wherein the target fragment comprises the end sequence of the first DNA fragment. According to an embodiment of the present invention, 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.

 According to still another aspect of the present invention, the present invention provides a method of constructing a DNA sequencing library. According to an embodiment of the present invention, 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. The inventors have surprisingly found that the use of this method to construct a DNA sequencing library is simple, rapid, efficient, and reproducible, and the resulting library is of very good quality. According to an embodiment of the present invention, 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.

According to another aspect of the invention, the invention provides a DNA sequencing library. According to an embodiment of the present invention, 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 In the embodiment, 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.

 According to still another aspect of the present invention, the present invention provides a method of DNA sequencing. According to an embodiment of the present invention, 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. The inventors have found that by using this method, 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.

 According to another aspect of the invention, the invention provides a method of determining genomic DNA sequence information. According to an embodiment of the present invention, 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.

 According to still another aspect of the present invention, the present invention provides an apparatus for preparing a DNA sample for sequencing. According to an embodiment of the present invention, 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, In order to obtain a fragment of interest, 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.

According to still another aspect of the present invention, the present invention provides a DNA sequencing system. According to an embodiment of the present invention, 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 . According to the invention For example, 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.

 The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS

 The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

 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. Detailed Description of the Invention

 The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the accompanying drawings, wherein the embodiments described in the drawings are intended to be illustrative only and not to limit the invention. .

 It should be noted that the terms "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.

Method of preparing a DNA sample for sequencing According to one aspect of the invention, 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:

 First, the genomic DNA is fragmented to obtain a first DNA fragment. According to an embodiment of the present invention, before the genomic DNA is fragmented, the step of extracting genomic DNA from the sample may be further included. According to an embodiment of the present invention, the source of the sample is not particularly limited. According to some specific examples of the invention, the sample may be derived from at least one of an animal, a plant, and a living organism. According to some embodiments of the invention, the animal may be at least one of a human and a penguin. According to an embodiment of the invention, the plant may be plum. According to an embodiment of the present invention, the method of fragmenting genomic DNA is not particularly limited. According to some specific examples of the present invention, 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. According to one embodiment of the invention, genomic DNA can be fragmented using a HydroShear instrument. According to an embodiment of the present invention, after obtaining the first DNA fragment, and before performing the end repair of the first DNA fragment, the method further comprises: performing segment selection on the first DNA fragment. According to an embodiment of the present invention, 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.

 Second, the first DNA fragment is end-repaired to obtain a DNA fragment that has been repaired at the end. According to an embodiment of the present invention, 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.

Next, the end-repaired DNA fragment is ligated to a capture marker to obtain a DNA fragment having a capture marker. The term "capture marker" as used herein 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. According to one embodiment of the present invention, 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) effectively capture and screen the DNA fragments labeled with biotin. In addition, 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. According to an embodiment of the present invention, 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. According to an embodiment of the present invention, after obtaining the DNA fragment having the capture marker, and before subjecting the DNA fragment having the capture marker to the cyclization treatment, it may further comprise: performing fragment selection on the DNA fragment having the capture marker. According to an embodiment of the present invention, 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. According to some embodiments of the invention, the DNA fragment having the capture marker may be 20-50 kb in length.

 Next, the DNA fragment having the capture marker is subjected to a cyclization treatment to obtain a circular DNA. According to an embodiment of the present invention, a DNA fragment having a marker can be subjected to a cyclization treatment using T4 DNA ligase and T3 DNA ligase. According to some embodiments of the present invention, after the DNA fragment having the label is subjected to a cyclization treatment, the step of removing the uncircularized DNA fragment may be further included. According to an embodiment of the present invention, the method of removing the uncircularized DNA fragment is not particularly limited. According to some specific examples of the present invention, the uncircularized DNA fragment can be removed by using at least one selected from the group consisting of a DNase and an exonuclease. According to an embodiment of the present invention, the DNase is an ATP-dependent DNase which does not degrade the plasmid, and the exonuclease is exonuclease I.

 Then, the circular DNA is fragmented to obtain a second DNA fragment. According to an embodiment of the invention, 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.

 Finally, the second DNA fragment is screened to obtain a fragment of interest which constitutes a DNA sample for sequencing. According to an embodiment of the present invention, the method of screening the second DNA fragment is not particularly limited. According to some embodiments of the invention, 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. According to an embodiment of the invention, the capture marker is a biotin and the molecular entity carried on the magnetic bead is streptavidin.

 According to an embodiment of the present invention, 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). Further based on high-throughput sequencing technology, it is possible to accurately determine partial sequence information of genomic DNA (in this context, the end sequence information of the first DNA fragment), thereby being effectively used for assisting genome assembly.

 DNA sequencing library and construction method thereof

 According to still another aspect of the present invention, 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:

 First, a method of preparing a DNA sample for sequencing according to an embodiment of the present invention, a target fragment is prepared.

Next, 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. According to an embodiment of the present invention, the method of performing end repair of the target fragment is not particularly limited. According to this issue In one embodiment, 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. According to an embodiment of the present invention, the method of adding the base A to the 3' end of the target fragment is not particularly limited. According to one embodiment of the present invention, Klenow (3 '-5' exo-) can be used to carry out the 3' end of the target fragment to base A.

 Next, the target fragment to which the base A is added at the end is attached to a linker to obtain a ligation product. According to an embodiment of the present invention, the method of attaching the target fragment to which the terminal A is added to the terminal is not particularly limited. According to one embodiment of the present invention, the target fragment to which the terminal A is added at the end is linked to a linker by using T4 DNA ligase.

 Next, the ligation product is subjected to PCR amplification to obtain an amplification product.

 Then, the amplified product is isolated and purified, and the amplified product constitutes a DNA sequencing library. According to an embodiment of the present invention, the method of separating and purifying the amplification product is not particularly limited. According to one embodiment of the present invention, the amplified product can be isolated and purified by 2% agarose gel electrophoresis.

 With the method of constructing a DNA sequencing library according to an embodiment of the present invention, 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.

 Specifically, according to an embodiment of the present invention, the method of constructing a DNA sequencing library of the present invention may comprise the following steps:

1) Randomly interrupt the sample genomic DNA into a 20-50 kb DNA fragment;

 2) Steps A or B below:

 A. fill the two ends of the interrupted DNA fragment, add the capture marker, and then isolate the first DNA fragment of 20 _ 50 kb; or

 B. Isolation of the interrupted 20 - 50 kb DNA fragment, and then fill the two ends of the DNA fragment, and add Wengji;

 3) cyclizing the isolated DNA fragment to obtain a circular DNA, and removing the uncircularized DNA fragment;

4) breaking the circular DNA into a DNA fragment of 100 - 2000b;

 5) separating the DNA fragment with the capture marker from the DNA fragment obtained in step 4) to obtain a capture fragment; preferably, it may also include

 6) the capture fragment is end-filled;

 Preferably, it may also include

7) 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;

 Preferably, it may also include

 8) A step of PCR amplification of the ligation product obtained in the step 7).

 According to an embodiment of the present invention, in step 1), the genomic DNA can be interrupted into a 25 - 50 kb DNA fragment. According to a specific example of the present invention, 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. According to an embodiment of the invention, 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). According to one embodiment of the present invention, the sample genomic DNA can be human, penguin (eg, Pygoscelis adeliae), or plum (eg, wild) Plum, (Prunus mume) genomic DNA. According to an embodiment of the present invention, 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. Preferably, 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. According to an embodiment of the present invention, 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), Thus, the range of disrupted fragments of genomic DNA can be increased to 20 _ 50 kb.

 According to an embodiment of the present invention, in step 2), the separation 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. According to some embodiments of the invention, the capture label can be a biotin, and correspondingly, the separation in step 5) can be carried out by using magnetic beads with streptavidin. According to an embodiment of the present invention, step 2) and step 5) may also be carried out based on a binding system similar to the antibody-antigen reaction.

 Due to the physically broken DNA fragment, a 5' or 3' end projection may be formed, and end filling is required. Therefore, according to an embodiment of the present invention, in step 2), 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. Among them, 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.

According to an embodiment of the present invention, in 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. According to a specific example of the present invention, it is also possible to directly perform end-filling using a base labeled with biotin. The above methods for labeling end-filled DNA fragments with Biotin are well within the knowledge and skill of those skilled in the art.

 According to an embodiment of the present invention, in step 3), 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. According to some specific examples of the present invention, 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. , comparing the effects of T3 DNA ligase and T4 DNA ligase, T3 DNA ligase alone, and T4 DNA ligase alone for ligation, the results showed that 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.

 According to an embodiment of the present invention, in the step 3), preferably, 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. Specifically, according to an embodiment of the present invention, the incubation temperature may be 60-70 ° C, such as 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 ° C, and 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. According to one embodiment of the present invention, the DNA mixture was incubated at 65 ° C for 15 minutes and immediately after a water bath.

 In step 3), the unligated fragmented DNA needs to be removed, otherwise the sequencing of the paired-end library is affected. According to an embodiment of the present invention, the unligated fragmented DNA can be removed by a known method of digesting linear DNA. According to a specific example of the present invention, an unconjugated double-stranded or single-stranded DNA can be degraded using a plasmid-safe ATP-dependent DNase or an exonuclease I. , using a non-degrading plasmid, ATP-dependent DNase and Exonuclease I, to remove unlooped double-stranded or single-stranded DNA, thereby enabling more complete digestion Linear linear DNA and single-stranded linear DNA minimize the effects of uncircularized linear DNA on the library.

According to an embodiment of the present invention, 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). Data), while using the intermediate linker for cyclization, it is easy to cause the library to lose a certain end sequence due to the position of the break in the intermediate linker interval during the interruption process, and the paired end cannot be formed, which limits the richness of the data. According to the embodiment of the present invention, 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).

 In step 4), since the circular DNA cannot be directly used for sequencing, it needs to be restored to linear DNA by fragmentation, and the paired terminal sequence is released. According to an embodiment of the present invention, 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.

 According to an embodiment of the present invention, in step 4), the circular DNA 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. In still another embodiment of the invention, the segment of 200-700b can be interrupted. According to still another embodiment of the present invention, 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.

 In 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. According to an embodiment of the present invention, after the base A is added, 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. In accordance with embodiments of the present invention, the linker can be selected from Illumina, SOLiD or 454 sequencing adaptors to accommodate sequencing sequencing of different sequencing platforms. According to an embodiment of the invention, the paired end fragments can be enriched by specific primers after ligation of the linker, which form a sequencing library. According to an embodiment of the present invention, 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.

According to another aspect of the invention, the invention provides a DNA sequencing library. According to an embodiment of the invention, 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.

 DNA sequencing method and method for determining genomic DNA sequence information

 According to still another aspect of the present invention, the present invention provides a method of DNA sequencing. According to an embodiment of the invention, the method comprises:

 First, 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.

 Then, and sequencing the DNA sequencing library of genomic DNA to obtain sequencing results. According to an embodiment of the present invention, a method of sequencing a DNA sequencing library of genomic DNA is not particularly limited. According to a specific example of the invention, sequencing can be performed using a high throughput sequencing platform (also referred to as "high throughput sequencing technology"). According to some embodiments of the invention, 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. According to some specific examples of the present invention, 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, and 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. According to an embodiment of the present invention, the method may further comprise: determining partial sequence information of the genomic DNA based on the sequencing result.

 By using the method of DNA sequencing according to an embodiment of the present invention, 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.

 According to another aspect of the invention, the invention provides a method of determining genomic DNA sequence information. According to an embodiment of the invention, the method comprises the steps of:

 First, the genomic DNA is divided into a first genomic DNA sample and a second genomic DNA sample.

 Next, using the first 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.

 Next, using the second genomic DNA sample, 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.

 Then, 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

 According to still another aspect of the present invention, the present invention provides an apparatus for preparing a DNA sample for sequencing. According to an embodiment of the present invention, referring to FIG. 7, 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. According to an embodiment of the present invention, any device suitable for fragmenting genomic DNA can be used as the first fragmentation unit 100. According to one embodiment of the invention, 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. According to an embodiment of the invention, 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. According to an embodiment of the present invention, the screening unit 600 is provided with a magnetic bead carrying streptavidin. According to some embodiments of the present invention, 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.

According to still another aspect of the present invention, the present invention provides a DNA sequencing system. According to an embodiment of the present invention, referring to FIG. 8, a DNA sequencing system 10000 includes: a sample preparation device 1000, a library construction device 2000, and a sequencing device 3000. Among them, 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. According to an embodiment of the present invention, 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. According to some embodiments of the invention, 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. According to some embodiments of the present invention, 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, and the single molecule sequencing platform may be selected from Helicos. The company's real single-molecule sequencing platform, Pacific Biosciences' single-molecule real-time sequencing platform, and at least one of Oxford Nanopore Technologies' nanopore sequencing platforms.

 According to an embodiment of the present invention, 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.

 It should be noted that the method for preparing a DNA sample for sequencing according to an embodiment of the present invention and its application are completed by the inventor of the present application through arduous creative labor and optimization work. The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available, for example, from Illumina.

 Example 1: DNA sequencing library construction and sequencing of penguin genome

 1. Construction of a DNA sequencing library for penguin genome

 A DNA sequencing library was constructed using 50 μ§ of genomic DNA of Pygoscelis adeliae as a library sample. In this example, the DNA sequencing library is a 40-45 kb insert end-pair library.

 1) Randomly interrupt genomic DNA

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. As shown in Figure 2, the schematics of each lane and the sample loading were: Lane 1, molecular weight standard λ-Hind III digest (Takara, Cat. No. D3403A); 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. 1051 1 -012); Lane 7, speed parameter 15, cycle number 40 interrupting effect, loading volume 200ng Lane 8, the speed parameter is 15, the number of cycles is 30, the loading is 200ng; Lane 9, the molecular weight standard Low Range PFG Marker (NEB, item number M0350S); Lane 10, speed parameter 16 , interrupting effect with a cycle number of 40, load 200ng; lane 1 1 , speed parameter 16 , cycle number 30 interrupt Effect, loading volume 200 ng; Lane 12, molecular weight standard lkb DNA Extension Ladder (Invitrogen, Cat. No. 1051 1-012); Lane 13, original genomic DNA, loading 150 ng. As can be seen from Fig. 3, 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. Well, it is possible to efficiently obtain a 20-50 kb DNA fragment.

 The breaking parameter of the standard Hydroshear instrument was set to a speed of 15 and a cycle number of 30, and then the ΙΟΟμΙ 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. Mix well, then place at room temperature for 10 minutes to fully bind the DNA to the magnetic beads, then place the EP tube on a magnetic stand for 2 minutes to allow the magnetic beads to be fully adsorbed to the tube wall, remove the supernatant, and add 500 μl 70% Ethanol, reverse several times, then remove the supernatant, add 500μ1 70% ethanol, invert several times, remove the supernatant, then dry the tube at 37 ° C until the beads are dry, then add 200μ1 elution Resuspend the magnetic beads in the buffer (QIAGEN). Specifically, place the tube at room temperature for 10 minutes to fully dissolve the DNA in the elution buffer, then place the tube on the magnetic stand for 2 minutes. The supernatant was transferred to a new manifold, and the magnetic beads were resuspended in the original tube by adding 185 μl 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.

 2) End-filling and biotin labeling

 Take 385 μl of the DNA fragment obtained in the previous step, add 50 μl of 10×T4 polynucleotide kinase buffer, 8 μl of 25 mM dNTP, 25 μl of Τ4 DNA polymerase (3000 units/ml, Enzymatics, Beverly, MA., USA), 5μ1 Klenow polymerase (5000 units/ml, Enzymatics) and 25μ1 T4 polynucleotide kinase (10000 units/ml, Enzymatics), then incubated at 20 ° C for 30 minutes, the end of the DNA fragment obtained in the previous step The DNA fragment was filled in to obtain the end-filled DNA fragment, and then purified using Agencourt AM purified magnetic beads to obtain a 345 μl end-filled DNA fragment, and then 50 μl of 10χΤ4 polynucleotide kinase buffer, 50 μl was added thereto. Biotin-dNTP, 25μ1 Τ4 DNA polymerase (3000 units/ml, Enzymatics, Beverly, MA., USA), 5μ1 Klenow polymerase (5000 units/ml, Enzymatics) and 25μ1 Τ4 polynucleotide kinase (10000 units/ Ml, Enzymatics), incubation at 20 °C for 30 minutes to obtain the biotinylated product, ready for use.

3) Electrophoresis separation The biotin-labeled product obtained in the previous step was formulated into a 500 μl reaction system by adding 5 μl of 20% SDS and 50 μl of 10 bromophenol blue. After mixing, the mixture was incubated at 65 ° C for 10 minutes, and then placed on water for 3 minutes. It was electrophoresed using 0.6% Megebase agarose gel, specifically, pulsed field electrophoresis for 16 hours at a voltage of 3.5 V/CM, switch time of 1 - 10 s, and then using ethidium bromide (EB). After gel staining, a 40-45 kb fragment was cut under Darkreader to obtain a 40-45 kb biotin-labeled DNA fragment, which was then subjected to gel recovery using a QIAEX II purification kit 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. As shown in Figure 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 μ§; 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.

 4) Cyclization

 The 40 _ 45 kb biotin-labeled DNA fragment obtained in the previous step was cyclized according to the following procedure: 2000 μl 2 χ ligase buffer, ΙΟΟμΙ Τ4 DNA ligase was added to a solution of 1000 ng 40 - 45 kb biotin-labeled DNA fragment ( 400,000 units/ml, NEB), ΙΟΟμΙ Τ3 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 μl 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.

 Then, 5 μl lOO mM guanidine, 60 μl 10x non-degrading plasmid ATP-dependent DNase buffer, 25 μl non-degrading plasmid ATP-dependent DNase (10000 units) were added to each EP tube. /ml, Epicentre) and 15μ1 exonuclease I (20000 units/ml, NEB), 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 μl of 0.5 Μ 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.

 5) Fragmentation of 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 50μ1 elution buffer to obtain purified DNA fragments. , spare.

 6) Magnetic bead capture, end-repair of DNA bound to magnetic beads and addition of base A

 Take 20μ1 magnetic beads ® Μ-280 streptavidin magnetic beads (Dynabeads® M-280 Streptavidin magnetic beads,

Invitrogen) in a 1.5 ml non-stick Rase-Free 1.5 ml Microfuge Tube, Ambion, AM12450 non-stick tube), placed on a magnetic stand for 1 minute, remove the supernatant, and wash the beads twice with 50 μM Bead Binding Buffer. The pellet was carefully resuspended, and the tube was allowed to stand on a magnetic separator for 1 minute, and the supernatant was discarded. Repeat this step once. The beads were then resuspended in 50 μM magnetic beads in combination with buffer and used.

 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 μM. 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 μΐ 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 μM of elution buffer to the centrifuge tube to obtain a resuspended magnetic bead DNA solution for use.

 Add ΙΟμΙ 10x T4 polynucleotide kinase buffer, 1.6μ1 25ηιΜ to the centrifuge tube (1ΝΤΡ, 5μ1 Τ4 DNA polymerase (3000 units/ml, Enzymatics, Beverly, MA., USA), Ιμΐ Klenow polymerase (5000 units/ml, Enzymatics) and 5μ1 T4 polynucleotide kinase (10000 units/ml, Enzymatics), then incubated at 20 °C for 30 minutes to end-end the DNA 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, and the procedure was the same as above. Then, 32 μΐ of the elution buffer was added to the centrifuge tube to resuspend the magnetic beads. Transfer to a new non-stick tube, add 5μl lOxBlue buffer, ΙΟμΙ 1 mM dATP and 3μ1 Klenow (3 '-5' exo-), mix and incubate at 37 ° C for 30 minutes, Add the base A to the end. Then, the magnetic beads were purified on the magnetic separation rack with Magnetic Bead Wash Buffer I and Elution Buffer, as above. Then add 19 μl of elution buffer to the non-stick tube. Resuspend the beads and transfer to a new non-stick tube for later use.

 7) Add linker and PCR amplification

Add 25 μl of 2xRapid Ligation Buffer, Ιμΐ Illumina double-end linker oligonucleotide (Illumina PE Adapter Oligo), and 5μ1 T4 DNA ligase (600,000 units/mL, Enzymatics) to the above-mentioned spare non-stick tube, and place it in 20 Incubation was carried out for 15 minutes at ° C for sequencing ligation, and then the magnetic beads were purified on a magnetic separation rack with Magnetic Bead Wash Buffer I and Elution Buffer, as above. Then, add 23 μl of elution buffer to the non-stick tube, resuspend the magnetic beads, transfer to a 0.2 ml PCR tube, add 25 μl of Phusion DNA polymerase and upstream and downstream primers, mix, and then use the following reaction procedure. 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.

 3. Sequencing results and analysis

 After sequencing the DNA sequencing library of the penguin genome, 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. The results are shown in Fig. 4. Figure 4 shows the results of the insertion range verification of the paired end sequences obtained in this example on the penguin genome. As can be seen from Fig. 4, 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.

 Penguin genome assembly using SOAPdenovo software (see, for example, Li, R, et al. The Sequenc e and de novo assembly of the giant panda genome. Nature 463, 311-3317 (2010); Li, R, et al. De novo assembly of Human genomes with massively parallel short read sequencing. Genome Res. 20:265-272 (2010), the entire disclosure of which is incorporated herein by reference, The 40 kb end sequence information was assembled, and the results of the assembly were as follows: scaffold N50 was significantly increased to 7500 kb; and when the penguin genome assembly scaffold N50 reached 5000 k b, the insertion of the penguin genome obtained above was 40 kb of end sequence information. As a result of the assembly, the assembly was: The scaffold N50 was significantly increased to 12,000 kb.

In this paper, the term "contig N50" or "scaffold N50" is used to mean: 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. Example 2: Construction and sequencing of DNA sequencing library of plum genome

 Using the genomic DNA of plum blossom as a genomic DNA sample, 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).

 Sequencing results and analysis

 Using SOAPdenovo software, the sequencing results were compared to the plum genome sequence, and the distance span of the paired end sequences obtained by sequencing the library on the genome was verified. The results are shown in Fig. 5. Fig. 5 shows the result of the insertion range verification of the paired terminal sequence obtained in the present example on the plum genome. As can be seen from Fig. 5, 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. When the scaffold N50 reached 570 kb, 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

 Using human genomic DNA as a genomic DNA sample, 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.

 Sequencing results and analysis

 Using SOAPdenovo software, the sequencing results were compared to the human genome sequence, and the distance span of the paired end sequences obtained by sequencing the library on the genome was verified. The results are shown in Fig. 6. 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. As can be seen from Fig. 6, 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.

 Human genome assembly was performed using SOAPdenovo software. When the scaffold N50 reached 1000 kb, the insert of the human genome obtained above was used to assemble 40 kb of end sequence information, and the assembly result was as follows: scaffold N50 was significantly increased to 2000 kb. Industrial applicability

Method for preparing DNA sample for sequencing, method for constructing DNA sequencing library, DNA sequencing library, method for DNA sequencing, method for determining genomic DNA sequence information, device for preparing DNA sample for sequencing, and DNA sequencing system , 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. According to an embodiment of the present invention, 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. By sequencing the paired-end library of 20-50 kb insertion length constructed by the present invention, 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.

 Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of those details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

 In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

Claim

A method of preparing a DNA sample for sequencing, comprising the steps of:

 Fragmenting the genomic DNA to obtain a first DNA fragment;

 End-repairing the first DNA fragment to obtain a DNA fragment subjected to end repair; attaching the end-repaired DNA fragment to a capture marker to obtain a DNA fragment having a capture marker; and the DNA having the capture marker The fragment is subjected to a cyclization treatment to obtain a circular DNA;

 Fragmenting the circular DNA to obtain a second DNA fragment;

 The second DNA fragment is screened to obtain a fragment of interest, which constitutes a DNA sample for sequencing.

 2. The method of claim 1 further comprising the step of extracting genomic DNA from the sample.

 3. Method according to claim 2, characterized in that the sample is derived from at least one of animals, plants and microorganisms.

 4. The method according to claim 3, wherein the animal is at least one of a human and a penguin.

 5. The method according to claim 3, wherein the plant is plum.

 6. The method according to claim 1, wherein the fragmenting of the genomic DNA is carried out by at least one selected from the group consisting of an atomization method, a sonication method, a HydroShear, and a digestion treatment.

 7. Method according to claim 6, characterized in that the genomic DNA is fragmented using a HydroShear instrument.

 8. The method according to claim 1, wherein after obtaining the first DNA fragment and before performing the end repair of the first DNA fragment, the method further comprises: performing segment selection on the first DNA fragment .

 9. The method according to claim 1, wherein the first DNA fragment has a length of 20 to 50 kb.

10. The method according to claim 1, wherein the first DNA fragment has a length of 25-50 kb.

The method according to claim 1, wherein the first DNA fragment is subjected to end repair and ligated capture, and the Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase are used, wherein The Klenow fragment has 5'→3' polymerase activity and 3'→5' polymerase activity, but lacks 5'→3' exonuclease activity.

 12. The method of claim 1 wherein the capture marker is biotin.

13. The method according to claim 1, wherein after obtaining the DNA fragment having the capture marker, and before subjecting the DNA fragment having the capture marker to cyclization, further comprising: having the capture The labeled DNA fragment is subjected to fragment selection.

14. Method according to claim 12 or 13, characterized in that the fragment selection is carried out by 0.6% agarose electrophoresis.

 The method according to claim 1, wherein the DNA fragment having the capture label has a length of 20 to 50 kb.

 The method according to claim 1, wherein the DNA fragment having the label is subjected to a cyclization treatment using T4 DNA ligase and T3 DNA ligase.

 17. The method according to claim 1, wherein the step of cyclizing the DNA fragment having the label further comprises the step of removing the uncircularized DNA fragment.

 18. The method according to claim 1, wherein the uncircularized DNA fragment is removed by using at least one selected from the group consisting of a DNase and an exonuclease.

 The method according to claim 18, wherein the DNase is an ATP-dependent DNase that does not degrade a plasmid, and the exonuclease is exonuclease I.

 20. The method according to claim 1, wherein the fragmentation of the circular DNA is carried out by at least one selected from the group consisting of a misting method, a sonication method, a HydroShear, and a digestion treatment.

 21. The method of claim 1 wherein the circular DNA is fragmented using a Covaris ultrasonic interrupter.

 22. The method of claim 1 wherein said second DNA fragment is between 100 and 1000 bp in length.

23. The method according to claim 1, wherein the second DNA fragment is 200-800 bp.

24. The method according to claim 1, wherein the screening of the second DNA fragment is performed by magnetic bead capture, wherein the magnetic bead carries a molecular entity capable of specifically recognizing the capture marker .

 25. The method of claim 24, wherein the capture marker is biotin and the molecular entity carried on the magnetic bead is streptavidin.

 26. A method of constructing a DNA sequencing library, comprising the steps of:

 The method according to claim 1, wherein the target fragment is prepared;

 The target fragment is subjected to end repair and base A is added to the 3' end to obtain a target fragment in which the base A is added at the end;

 Adding the target fragment of the terminal A to the terminal to the linker to obtain a ligation product;

 The ligation product is subjected to PCR amplification to obtain an amplification product;

The amplification product is isolated and purified, and the amplification product constitutes the DNA sequencing library.

27. The method according to claim 26, wherein the end fragment repair of the fragment of interest is carried out using a Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase, wherein the Klenow fragment has 5' →3' polymerase activity and 3'→5' polymerase activity, but lacking 5'→3' exonuclease activity.

 The method according to claim 26, wherein the target fragment is subjected to addition of a base A at the 3' end using Klenow (3 '-5' exo-).

 The method according to claim 26, wherein the attachment of the target fragment to which the terminal A is added to the terminal is carried out using T4 DNA ligase.

 30. The method according to claim 26, wherein the amplification product is isolated and purified by 2% agarose gel electrophoresis.

 31. A DNA sequencing library constructed by the method of any one of claims 26-30.

32. A method of DNA sequencing, comprising:

 The method according to any one of claims 26 to 30, wherein a DNA sequencing library of genomic DNA is constructed; and the DNA sequencing library of the genomic DNA is sequenced to obtain a sequencing result.

 33. The method of claim 32, wherein said sequencing is performed using a high throughput sequencing platform.

 34. The method of claim 32, wherein the sequencing is performed using at least one selected from the group consisting of a second generation sequencing platform and a single molecule sequencing platform.

 35. The method according to claim 34, wherein the second generation sequencing platform is 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 The molecular sequencing platform is at least one of a real single molecule sequencing platform selected from Helicos, a single molecule real-time sequencing platform from Pacific Biosciences, and a nanopore sequencing platform from Oxford Nanopore Technologies.

 36. The method of claim 34, further comprising:

 Based on the sequencing results, partial sequence information of the genomic DNA is determined.

 37. A method of determining genomic DNA sequence information, comprising the steps of:

 The genomic DNA is divided into a first genomic DNA sample and a second genomic DNA sample;

 Using the first genomic DNA sample, sequencing the first genomic DNA sample by the method according to any one of claims 32-36, and determining partial sequence information of the genomic DNA based on the sequencing result; The second genomic DNA sample is subjected to sequencing according to a conventional sequencing method to obtain sequencing data of the genomic DNA, wherein the conventional sequencing method is selected from the group consisting of SOLEXA, SOLID, 454, And at least one of single molecule sequencing techniques;

The partial sequence information of the genomic DNA is assembled and spliced with the sequencing data of the genomic DNA to determine sequence information of the genomic DNA.

38. A device for preparing a DNA sample for sequencing, comprising:

 a first fragmentation unit for fragmenting genomic DNA to obtain a first DNA fragment;

 An end repairing unit, the end repairing unit is coupled to the first fragmentation unit for performing end repair of the first DNA fragment to obtain a DNA fragment that has been repaired at the end;

 a labeling unit, the labeling unit being connected to the end repairing unit, configured to connect the end-repaired DNA fragment to a capture marker to obtain a DNA fragment having a capture marker;

 a cyclization unit, the cyclization unit is coupled to the labeling unit, and configured to cyclize the DNA fragment having the capture label to obtain a circular DNA;

 a second fragmentation unit, the second fragmentation unit being ligated to the cyclization unit for fragmenting the circular DNA to obtain a second DNA fragment;

 a screening unit, the screening unit being coupled to the second fragmentation unit for screening the second DNA fragment to obtain a target fragment, the target fragment constituting a DNA sample for sequencing.

 39. The device according to claim 38, further comprising:

 A genome extraction unit, the genome extraction unit being coupled to the first fragmentation unit for extracting genomic DNA from the biological sample.

 40. Apparatus according to claim 38 wherein said first fragmentation unit is a HydroShear instrument.

 41. Apparatus according to claim 38 wherein said second fragmentation unit is a Covaris ultrasonic interrupter.

 42. The device according to claim 38, wherein biotin is provided in the marking unit.

 43. The apparatus according to claim 42, wherein the screening unit is provided with a magnetic bead carrying streptavidin.

 44. A DNA sequencing system, comprising:

 A sample preparation device, which is the device for preparing a DNA sample for sequencing according to any one of claims 38 to 43 for preparing a fragment of interest, the fragment of interest constituting a DNA sample for sequencing;

 a library construction device, the library construction device being coupled to the sample preparation device for constructing a sequencing library for the DNA sample for sequencing;

 A sequencing device is coupled to the library construction device for sequencing the sequencing library.

45. The system according to claim 44, wherein the library construction device further comprises: an end modification unit, wherein the end modification unit is configured to connect the target segment to a linker to obtain a connection product. Object

 a PCR amplification unit, the PCR amplification unit being linked to the terminal modification unit for amplifying the ligation product to obtain an amplification product;

 And a purification unit, wherein the purification unit is coupled to the PCR amplification unit for isolating and purifying the amplification product, and the amplification product constitutes the DNA sequencing library.

 46. The system of claim 44, wherein the sequencing device is at least one selected from the group consisting of a second generation sequencing platform and a single molecule sequencing platform.

 47. The system of claim 46, wherein the second generation sequencing platform is 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.

 48. The system of claim 46, wherein the single molecule sequencing platform is a real single molecule sequencing platform selected from Helicos, a single molecule real time sequencing platform from Pacific Biosciences, and a nanometer from Oxford Nanopore Technologies. At least one of the sequencing platforms.