KR101632881B1 - Sequencing method of genomic DNA end sequence using NGS - Google Patents

Abstract

The present invention relates to a method for analyzing a base sequence of dielectric DNA by using NGS after amplifying only a large quantity of terminal base sequences of dielectric DNA by using a primer which is specific to a dielectric DNA terminal base sequence. Terminal base sequence information of dielectric DNA inserted in a vector may be rapidly, conveniently, and cheaply mass-produced. A sequence of contigs or scaffolds may be easily determined by using a large quantity of analyzed terminal base sequence information. An ultra-large scaffold having tens of mega size may be constructed, and a precise dielectric physical map may be written. A position of a clone corresponding to analyzed terminal base sequence information may be clarified, so the method according to the present invention may be used in enhancing the quality of dielectric analysis such as sequence analysis gap connection and sequence accuracy verification.

Description

[0001] The present invention relates to a method for mass analysis of genomic DNA end sequence using a next-generation sequencing method (Sequencing method of genomic DNA end sequence using NGS)

The present invention relates to a method for analyzing a genomic DNA sequence using NGS. More specifically, a primer specific to a DNA base sequence is amplified to amplify a large amount of a base sequence of a genomic DNA, To a method for analyzing a nucleotide sequence.

The Next Generation Sequencing (NGS) technology, which is currently the mainstream genome sequencing method, is a technique that genomic DNA extracted from a cell is fragmented to rapidly decode a fragment of the fragmented DNA fragment in a short time, The algorithm is used to reconstruct the decoded DNA fragments of several billion DNA fragments to complete the original genome structure. However, this method is disadvantageous in that the nucleotide sequence of the recombinant genome information (scaffold, contig) and the structure of the genome produced by the NGS device itself due to the DNA decoding error and the computer assembling program error are inferior to the conventional method .

Most of the NGS models (Illumina, Roche, LifeTechnology) sculpt the genomic DNA to a size of 3kb, 5kb, 10kb and so on, (Mate-pair sequencing), and then uses the nucleotide sequence information and the size of the fragment as a bridge sequence information to recombine the DNA. Meanwhile, Pacific Bio decodes relatively long DNA fragments of 10 kb to 20 kb at a time and uses them for data assembling.

The use of bridge clone sequences to create contigs and scaffolds is similar to the 'genome physical map' technique traditionally used in the human genome project. Dielectric physical maps are a method of aligning sequences of sequenced conics and scaffolds. They improve the accuracy of dielectric structures, such as 'sequence gap connection', 'sequence accuracy' verification, and large scale scaffold construction It is used as a very important technology.

The core technology used in genome mapping is to utilize the information of inserted genomic DNA ends by propagating the genomic DNA of the study using a self-propagating plasmid, a vector, in E. coli. In general, the types of vectors are divided into BAC (Bacterial Artificial Chromosome), Fosme, Cosmid and the like according to the size of insertable DNA in the vector.

For example, in the case of BAC vectors, genomic DNA insertion of 100,000 to 200,000 bases is usually possible. As shown in FIG. 1, the BAC clone into which the genomic DNA is inserted is composed of two regions, a BAC vector and a genomic DNA inserted therein. The vector-specific base sequence (T7 promoter and SP6 promoter) Lt; RTI ID = 0.0 > DNA < / RTI > Therefore, it is possible to decode the base sequence of both ends of the inserted genomic DNA by using these T7 promoter and SP6 promoter as a sequencing primer. The direction of both terminal sequences of the inserted genomic DNA is distinguished according to the type of the promoter used for the sequence analysis. The terminus of the genomic DNA decoded by the T7 promoter is referred to as' T7-BAC end sequence (T7-BAC end sequence, T7-BES ), And the end of the genomic DNA terminal sequence decoded by the SP6 promoter is called 'SP6-BAC end sequence (SP6-BES)'. These 'T7-BES' and 'SP6-BES' are indispensable information for 'Genome Physics Map' and have traditionally been used to verify genome structure and sequence analysis errors in many genome detoxification studies including the Human Genome Project.

Methods for decoding BES of genomic DNA inserted in BAC clones include the traditional first-generation capillary sequencing method and the NGS method. First-generation gene analysis is a method of sequencing BAC clones one by one. Since BAC clones can be located in accordance with the decoded BES information, it is possible to use them as a material in subsequent studies. It takes a long time and the data production cost is very expensive. In recent years, it is possible to decode BES base using NGS device, which collects tens of thousands of BAC clones into NGS sequencing library (NXSeq library) to decode in large quantities. However, this method is not only low in efficiency because it is possible to mass-decipher, but the amount of 'T7-BES' and 'SP6-BES' finally obtained is small and corresponds to the nucleotide sequence information of the BAC clone after detoxification which is the greatest advantage of BES sequence analysis The BAC clones can not be used for subsequent studies because it is impossible to identify the location of the BAC clones.

1. Korean Patent Registration No. 10-1406720 2. Korean Patent Registration No. 10-1447593 3. Korean Patent Registration No. 10-1533792 4. Korean Patent Publication No. 10-2015-0017525

In order to solve the above problems, the present invention provides a method for rapidly and cheaply disassembling the terminal nucleotide sequence of a genomic DNA inserted into a vector by the NGS method using a primer capable of specifically amplifying only the terminal base sequence (BES) of the genomic DNA And a method for identifying the position of a clone corresponding to the produced BES information.

In order to achieve the above object, the present invention provides a method for preparing a cell stock comprising the steps of: preparing a BAC library by extracting genomic DNA to be analyzed; collecting each BAC clone in a 384-well plate to prepare a cell stock; Three-dimensional BAC clones of the 384-well plate for cell stock are three-dimensionally combined to prepare a three-dimensional BAC library consisting of 60 stores; Amplifying the BAC clones of the 3-dimensional BAC library using an Escherichia coli culture and extracting BAC clone DNA; Cutting the extracted BAC clone DNA to an appropriate size necessary for analysis; Attaching a Y-type adapter primer to both ends of the fragment after smoothing both ends of the DNA fragment; Amplifying both terminal sequences of the genomic DNA using primers comprising the SP6 promoter sequence of the BAC vector or a primer comprising the T7 promoter sequence; Purifying the amplified DNA and fractionating it into a size suitable for NGS sequence analysis; Amplifying the fractionated DNA by emPCR; Analyzing the sequence of the amplified DNA with NGS to obtain sequence data; And identifying the position of the BAC clone using the sequence data. The present invention also provides a method for mass analysis of a genomic DNA end sequence using a next-generation sequencing method.

In the above method, the 384-well plate for cell stock is coated with a marker (in a row, column and plate) so as to distinguish the origins of the genomic DNA collected on the X axis (horizontal), Y axis It is desirable to number them.

In the method, the three-dimensional BAC library comprises:

Collecting 24 BAC clones on the horizontal axis (X axis) corresponding to the same number in 384-well plates for cell stock in one reservoir; Collecting 16 BAC clones in the vertical axis (Y axis) corresponding to the same number in a 384-well plate for cell stock into one reservoir; And collecting 384 BAC clones contained in one 384-well plate for cell stock into one reservoir to obtain a plate pool (Z axis).

In this method, it is preferable that the Y-type adapter primer has the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, and the terminal nucleotide sequence of the five nucleotide sequences is the non-complementary primer.

In the above method, the extracted BAC clone DNA is preferably cut using ultrasonic waves.

In this method, the primer comprising the SP6 promoter sequence is preferably a GSB-SP6 primer consisting of the forward adapter of SEQ ID NO: 3, the bar code sequence representing the position in the 3D BAC library, and the SP6 promoter of SEQ ID NO: 4.

In this method, the primer comprising the T7 promoter sequence comprises a forward adapter of SEQ ID NO: 5, a barcode sequence representing the position in the 3D BAC library, and GSB-T7 comprising the T7 promoter of SEQ ID NO: 6 Primer is preferred.

In this method, the amplification of both end sequences of the genomic DNA is carried out using the GSB-SP6 primer of claim 6 and the GSB-RP primer of SEQ ID NO: 7, or the GSB-T7 primer of claim 7 and the GSB-RP primer of SEQ ID NO: .

The method of the present invention can mass-amplify both terminal base sequences of a genomic DNA to be analyzed and mass-decode it using an NGS instrument and quickly locate the BAC clone corresponding to each terminal base sequence, It is possible to mass-produce the DNA base sequence information quickly, simply and inexpensively.

In comparison with the 7,680 clones performed in the examples of the present invention, the conventional first-generation gene analysis technology (based on one ABI3730XL) takes about one month or more, whereas the method of the present invention allows analysis in one day Cost of less than 1/10 of first-generation gene analysis technology. In addition, in the first generation gene analysis technique, since the preprocessing step of the sequence analysis is very complicated, several manpower is required, but the method of the present invention is simple enough to carry out the entire process.

In addition, it is possible to easily determine the sequence of conic or scaffold using the decoded large amount of terminal sequence information, and it is possible to make precise dielectric physical mapping such as the construction of a very large scaffold of several tens of mega size.

In addition, the method of the present invention can be used to improve the quality of genome detoxification such as sequencing gap closing and sequence accuracy verification because the location of the clone corresponding to the decoded terminal sequence information can be identified.

Figure 1 shows the structure of a BAC clone.
2 is an overall configuration diagram of the present invention.
3 is a schematic diagram of a three-dimensional BAC library production.
Figure 4 shows the extraction results of BAC clone DNA.
Figure 5 shows fragmentation results of BAC clone DNA.
6 shows the sequence information of GSB-YAP primer and the underlined region shows the non-complementary base sequence.
7 is an adapter primer sequence used for amplification of a DNA base sequence inserted into a BAC vector.
Figure 8 shows the results of PCR using GSB-SP6 / GSB-RP and GSB-T7 / GSB-RP primers.
Figure 9 shows the size fraction of PCR DNA.
10 is a nucleotide sequence structure of sequence reads fatigued with GS-FLX.
11 is a diagram showing the structure of sequence data analysis.
Figure 12 is an example of genetic physical mapping using the BAC end sequence.

In the present invention, BAC is used as a vector, but all commonly used vectors can be used.

The overall configuration of the method of the present invention is as shown in FIG. 2, and details of each step are as follows.

1. Cells Of stock  making

The BAC library is prepared by extracting the genomic DNA of the subject to be analyzed, and each BAC clone is collected in a 384-well plate to prepare a cell stock.

2. 3D BAC  Building the Library

A three-dimensional BAC library consisting of 60 repositories of 3-dimentional pooling (3D-pool) is prepared by combining 20 BAC clones of the 384-well plate for cell stocks three-dimensionally. The three-dimensional BAC library was obtained by extracting each BAC clone stored in a 384-well plate by a predetermined amount on each of a horizontal axis (X axis), a vertical axis (Y axis), and a plate pool (Z axis) , And a library in which each BAC clone collected in the Z axis is mixed into one reservoir.

The concrete procedure for producing the 3D BAC library is as follows.

(1) Collect the 24 BAC clones on the horizontal axis (X-axis) corresponding to the same number in one store in 384-well plate for cell stock.

(2) Collect 16 BAC clones in vertical (Y axis) corresponding to the same number in 384-well plates for cell stock into one reservoir.

(3) Collect 384 BAC clones contained in a 384-well plate for cell stock in one reservoir to obtain a plate pool (Z axis).

Preferably, the 384-well plate for cell stock is provided with marker numbers in a row, column, and plate unit so as to distinguish the origins of the genomic DNA collected on the X axis, the Y axis, and the Z axis. In detail, 16 alphabets (A to P) are placed on the upper side of the 384-well plate with 24 Arabic numerals (1 to 24) on the horizontal axis (X axis) and a vertical axis BAC clones corresponding to the same X-axis or Y-axis sequence in each of the plates to be desired were picked out in a predetermined amount and placed in each container according to each row. The Z axis was measured in 384 BAC clones contained in one 384- And extracted into a single container to prepare a 3D BAC library.

3. BAC  The proliferation of clones and BAC  Clone DNA  extraction

BAC clones of the 3-dimensional BAC library are amplified using a culture medium for culturing E. coli, and then BAC clone DNA is extracted.

4. Extracted DNA Cutting of

The extracted BAC clone DNA is cut to an appropriate size for analysis. At this time, various methods commonly used can be used for cutting the DNA, but it is more preferable to cut the DNA using ultrasonic waves.

5. Y-type adapter Primer (Y-type adapter primer)  Attach

Both ends of the cleaved DNA fragment are blunt-ended, and a Y-type adapter primer is attached to both ends of the fragment so that DNA fragments can not bind to each other. The Y-type adapter primer has a non-complementary sequence at its terminus to prevent self-assembly of the genomic DNA during amplification of the DNA end sequence.

An example of a Y-type adapter primer is shown in SEQ ID NO: 1, SEQ ID NO: 2, and FIG. 6 of the Sequence Listing. As shown in Fig. 6, the five terminal sequences are non-complementary to each other.

6. Dielectric DNA  Amplification of both end sequences

Both ends of the genomic DNA are amplified by PCR using the SP6 promoter and the T7 promoter sequence inherent in the BAC vector.

It is preferable to amplify both terminal sequences of the genomic DNA using a primer comprising the SP6 promoter sequence or a primer comprising the T7 promoter sequence.

The GSB-SP6 primer, which is an example of a primer containing the SP6 promoter sequence, consists of the forward adapter (30 bp) of SEQ ID NO: 3, the bar code sequence (10 bp) and the SP6 promoter of SEQ ID NO: 4 (18 bp). The GSB-T7 primer, which is an example of a primer containing the T7 promoter sequence, consists of the forward adapter (30 bp) of SEQ ID NO: 5, the bar code sequence (10 bp) and the T7 promoter of SEQ ID NO: 6 (20 bp). The GSB-RP primer, which is an example of a reverse primer used together with the primer, has the sequence of SEQ ID NO: 7. At this time, the barcode sequence is a marker sequence indicating the position in the 3-dimensional BAC library. The construction of the primers is shown in FIG.

PCR is performed by dividing the DNA of one reservoir into two and adding GSB-SP6 primer and GSB-RP primer, and the other one is preferably performing PCR by inserting GSB-T7 primer and GSB-RP primer.

7. Amplified DNA Purification and size fractionation

The amplified DNA is purified and fractionated to a size suitable for NGS sequencing. For example, when using a GS-FLX sequencer for NGS analysis, the average size is preferably 700 bp.

8. emPCR On by DNA Amplification of

The DNA fragments of the appropriate size are amplified by emPCR. PCR amplification was carried out using dNTP, PCR buffer, primer, Taq polymerase, and PPiase (Peptidyl-Prolyl Cis-Trans Isomerase) / RTI >

9. NGS ≪ / RTI >

Sequence of the amplified DNA is analyzed by NGS to obtain sequence data.

10. BAC  Location of the clone

Using the sequence data obtained from the NGS sequence analysis, the location of the BAC clone is identified.

In the obtained sequence data, the adapter primer at the 3'-terminal region is removed and the sequence data with the adapter primer at the 5'-terminal region are obtained. The obtained sequence data is divided into 60 types using the barcode sequence used for the production of the three-dimensional sequence library, and the sequence data collected for each barcode is conic. Homologies between the cones corresponding to the X axis, the Y axis and the Z axis are searched and classified by the same sequence, and the position of the SP6 promoter or T7 promoter is reclassified by the same clone to identify the position of the BAC clone.

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not limited by these examples.

< Example  1>

cell Stock  making

After extracting the Tenebrio genomic DNA, a BAC library was prepared using the CopyRight v2.0 BAC Cloning Kit (Lucigen) and each BAC clone was picked into a 384-well plate to make a cell stock. The resulting cell stock was stored in a -80 ° C freezer.

< Example  2>

3D BAC  The library (3D- dimentional BAC library Production

A three-dimensional BAC library consisting of 60 reservoirs was prepared by mixing 20 384-well plates, cell stock prepared by the above method. The production process of the 3-dimensional BAC library is shown in FIG. 3, and a concrete production method is as follows.

(1) 20 sheets of 384-wells stored as cell stocks in a -80 DEG C freezer were transferred to a 4 DEG C refrigerator and thawed.

(2) The 384-well plate is a structure in which 16 (× A to × P) wells are arranged in 24 ('× 1' to '× 24') and vertical (Y-axis) , 7,680 BAC clones (384 clones / plate x 20 plates) stored in 20 384 transparent well plates were used in the present invention.

(16 wells) of the # 1 plate, the h1 lane (16 wells) of the # 2 plate, the h1 lane (16 wells) of the # 20 plate, (Rx1 'to Rx24') were prepared from the X-axis by mixing stocks (Rx) containing 50 ml of 1 × LB medium. In the same manner, 16 mixtures ('RyA' to 'RyP') were prepared from the Y axis, and a plate pool (Z axis) in which 384 cells of each plate were mixed in one reservoir (X-axis: 24, Y-axis: 16, and Z-axis: 20) from 7,680 BAC clones.

(4) The BAC clone mixture collected in each reservoir was mixed with an equal amount of DMSO frozen stock solution and stored in a -80 ° C freezer.

(5) Each of 312 占 퐇 (X axis), 208 占 퐇 (Y axis) and 500 占 퐇 (Z axis) were taken from a storage stock of a -80 占 폚 freezer and mixed with 50 ml of 1 占 LB medium. And incubated for 6 hours with shaking at 200 rpm (OD = about 1.0).

< Example  3>

BAC  Clone DNA Extraction of

BAC clone DNA was extracted using HiPure plasmid kit (Invitrogen). 4 ml of a suspension buffer, 4 ml of a lysis buffer and 4 ml of a neutralizing buffer were sequentially added to the cells collected by the centrifugation method in accordance with the use method described in the kit, The cells were reacted for a certain period of time to finally dissolve cell membranes. The supernatant obtained by centrifugation of the dissolved liquid was placed in a column, allowed to stand at room temperature for a certain time, and then passed through a column to extract BAC clone DNA.

The results of extraction of BAC clone DNA are shown in Fig. In FIG. 4, 1 to 6 lanes are obtained by loading 1 각 each of BAC clone DNA (= 100 to 120 ng), and M is 1 kb lambda ladder.

The amount of BAC clone DNA extracted from a total of 60 reservoirs was 1 to 4 μg each.

< Example  4>

DNA Fragmentation

1 μg of BAC clone DNA extracted into a 1.5 ml microtube was added and the volume was adjusted to 1 μl with a total volume of 50 μl. The volume of the high (high) mode of US portable cleaners (US-05, JEIOTECH, Korea) mode) for 30 to 120 seconds to fragment the DNA to a size of 100 bp to 10 kb. The results are shown in Fig. (= 100 to 120 ng), respectively, and the size marker is a 100 bp ladder.

< Example  5>

Fragmented DNA  Restoration of distal site

The end repairing enzyme mix kit (Fermentas, K0771) was used to blunt the end of the fragmented DNA.

The buffer and enzyme were mixed in a 1.5 ml microtube containing each DNA according to the manufacturer's kit method, and the mixture was reacted in a water bath block at 20 ° C for 5 minutes. Then, phenol chloroform and ethanol precipitation The DNA was purified by the method and the DNA was dissolved in 6 sterilized water and its concentration was measured.

< Example  6>

Y-type adapter Primer  Combination

Y-type adapter primer (GSB-YAP) was attached to both ends to prevent self-ligation of DNA fragments as a primer during the polymerase reaction.

The GSB-YAP used was 18 bp double-stranded DNA. It has a structure in which the 5 nucleotide sequences at the terminal region are not complementary to each other. The GSB-YAP primer sequence information is shown in FIG. 6, and the underlined region in FIG. 6 represents the non-complementary base sequence.

To the tube containing 200 ng of smooth-polished DNA and 200 ng of GSB-YAP, 2.5 리 of ligase was mixed and reacted in a water bath at 16 캜 for 3 hours. After the reaction was completed, the DNA was purified using a DNA purification kit, and the DNA was dissolved in 30 μl of sterilized water.

< Example  7>

BAC  Dielectric inserted into vector DNA  Amplification of the terminal base sequence

SPB promoter or T7 promoter sequence, a barcode sequence, a forward adapter primer for emPCR (forward primer) (GSB-SP6 and Bacillus spp.) In order to specifically amplify both terminal base sequences of the inserted genomic DNA GSB-T7) were designed and manufactured. The sequences of GSB-SP6 primer (SEQ ID NO: 2), GSB-T7 primer (SEQ ID NO: 3) and GSB_RP primer (SEQ ID NO: 4) which are the primers used for amplification of the DNA base sequence inserted into the BAC vector are shown in FIG. .

GSB-SP6 primer and GSB-RP primer were inserted into one tube, and GSB-T7 primer and GSB-RP adapter primer were added to the other tube to perform PCR.

PCR amplification was carried out using a ProDNi thermocycler (GnC Bio). Each reaction was performed by adding 0.25 μl of Taq DNA polymerase, 2.5 μl of 10 × buffer, 0.5 μl of 50 × dNTP, 10 pmol of SP6 primer, 1 mu l / 10 pmol of GSB-RP primer and 3 mu l of template DNA (Template DNA). The reaction was denatured at 96 캜 for 3 minutes, repeated 40 times at 96 캜 for 20 seconds, 50 캜 for 20 seconds, and 72 캜 for 20 seconds, followed by reaction at 72 캜 for 10 minutes. After the amplification reaction, electrophoresis was performed on agarose gel at a concentration of 1%, followed by coloring with ethidium bromide and confirmed in a UV transilluminator. The results are shown in Fig. 8, M is a 100 bp size marker.

As a result, it was confirmed that the PCR fragment of 2.0 kb or less was amplified in FIG. 8 (a), and PCR was performed on BAC clones of 60 reservoirs collected on the X axis, Y axis and Z axis, b.

< Example  8>

Size fraction ( Size fraction )

120 DNAs amplified with 60 types of GSB-T7 / GSB-RP and 60 types of GSB-SP6 / GSB-RP were reassembled into a single tube, and then the average sizes of GS-FLX sequencers To extract 700 bp of DNA, size fractions were performed with a chroma spin TE1000 column kit (Clontech TE1000 column kit, Clontech) and Ampure bead (Beckmancoulter). The kit was used according to the product manual.

Approximately 100 DNA of DNA was passed through a TE1000 column containing size fraction beads and freely dropped. Ten total droplets were collected, and the size was confirmed by electrophoresis (Fig. 9 (a)). The second step was performed with the DNA of the second drop.

To remove small size DNA, 250 μl of Ampou XP Bead was added to a tube containing the DNA of the 10th drop from which large size DNA had been removed, and the DNA was mounted on the MPC apparatus. Then, the buffer was removed, (sizing solution) was added and mixed with amphire XP beads. 125 [mu] l of the sizing mix was collected and re-blended with 50 [mu] l of the 10th PCR product, and then incubated for 5 minutes in a water bath at 25 [deg.] C and mounted on MPC to recover the supernatant. 125 쨉 l of the recovered supernatant was added to 375 쨉 l of the remaining sizing mixture, and the mixture was incubated in a 25 째 C water bath, and then mounted in MPC to remove supernatant. 100 쨉 l of TE buffer was added to the tube from which the supernatant was removed, and vortexed. Then, 500 쨉 l of a sizing solution was added, followed by shaking, followed by incubation in a water bath at 25 캜 for 5 minutes. This solution was mounted on MPC, the supernatant was removed, washed twice with 70% ethanol, and then the amphora XP beads were air-dried. 23 쨉 l of TE buffer was added to the dried beads, followed by shaking, and then mounted on MPC to recover 21 쨉 l of the supernatant. The size and concentration of the recovered DNA were measured with a BioAnalyzer 2100 (Bioanalyzer 2100, BECKMAN Co.) using a pico RNA chip. As a result, it was confirmed that the DNA concentration was 2.24 ng / μl and the average size was 704 bp (FIG. 9 b)

< Example  9>

emPCR through DNA Amplification of

The reagents and methods required for the entire process were performed according to the Roche method. A premix prepared by mixing dNTP, PCR buffer, primer, Taq polymerase and PPiase was dispensed into 32 emulsion tubes and recovered in Example 8 to obtain a bioanalyzer PicoRNA GSB- RPnning chip) and the number of beads were mixed in an appropriate amount. Then, PCR was performed to lower the temperature sequentially from 80 ° C to 20 ° C so that the beads and single-stranded DNA could bind. Sterile water was added to the DNA-captured beads after the reaction, and the PCR premix was mixed with the mixed oil and shaken at 12 Hz for 5 minutes using a Tissue lyser PCR oil molecules (microreactors) were made. A tube containing the PCR oil molecule was attached to the PCR instrument and subjected to PCR to amplify the mixed DNA in the oil molecule.

< Example  10>

GS - FLX  Sequencing

The reagents and methods required for the entire process were performed according to the Roche method. Streptavidin-coated beads were recovered from the emPCR-treated samples using isopropanol, ethanol, enhancing fluid buffer and the like. After the beads were neutralized with a melting solution and an annealing buffer, enrichment primers were added and reacted for 5 minutes in a water bath at 65 ° C. to prepare a sequence analysis sample. The enrichment beads, which had been washed several times with an enhancing buffer, were mixed with the prepared DNA samples and washed with a molten solution. Only the beads bound to the target DNA were recovered by the sequencing primer. The recovered capture beads were placed in a PicoTiter ^TM plate and subjected to pyrosequencing for 12 hours. The structure of the finally obtained sequence is shown in Fig. Each sequence reading can be classified as an SP6 promoter, a T7 promoter, and a barcode sequence.

< Example  11>

Sequence data ( Sequence data ) analysis

The adapter sequence (GSB-1) attached to the 3'-end of the sequence library construct by the BlastN method after obtaining 647,759 sequencing readings by performing the fat-sequencing with the XL70 sequencing library kit, RP) were removed, and 171,104 and 234,984 readings of sequence analysis with GSB-SP6 and GSB-T7 attached to the 5 'end region with adapter primer, respectively, were obtained. Each of the obtained sequencing analyzes was separated again into 60 types (X-axis: 24, Y-axis: 16, Z-axis: 20) using the bar code sequence used for the 3D-pool library, Sequence analysis read by bar code was made conic using CAP3 assembler. The homology search was performed among the cones corresponding to each of the X axis, Y axis and Z axis, and classified according to the same sequence. Then, SP6 and T7 were reclassified by identical clones to finally locate the BAC clone. Fig. 11 shows the sequence data analysis structure.

As a result of cross-searching of sequence homology using X-axis, Y-axis and Z-axis using BlastN, the number of BAC clones in which clones were found in 7,680 kinds (384-well plates 20) 7,108 (93% of the experimental group). The sequence length of each reading was 17bp ~ 793bp, and the mean length was 268bp. Of the 7,108 sites identified, 5,062 clones (66% of the experimental group), 1,153 clones (15% of the experimental group), and only T7 were identified with both end sequences SP6 and T7 of the BAC clones The clones were 893 (12%).

< Example  12>

BAC  Dielectric Physical Map Using Terminal Sequence genome physical map ) write

The BAC end sequence obtained in the present invention was subjected to homology search using the BLASTN method for the conic and scaffold sequences obtained by shot gun sequence analysis and assembly using NGS instruments such as HiSeq 2500 and GS-FLX, A map was created and the results are shown in Fig. We could identify the sequence of scaffolds according to the genome structure by using both terminal sequences (SP6 for black and T7 for red) of the identified BACs.

<110> GnC Bio Co., LTD. <120> Sequencing method of genomic DNA end sequence using NGS <130> 000 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GSB-YAP primer <400> 1 ccgatctaga tcgtccga 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GSB-YAP primer <400> 2 ggctagatct agcgtcct 18 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward adapter in GSB-SP6 primer <400> 3 ccatctcatc cctgcgtgtc tccgactcag 30 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SP6 promoter in GSB-SP6 primer <400> 4 atttaggtga cactatag 18 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward adapter in GSB-T7 primer <400> 5 ccatctcatc cctgcgtgtc tccgactcag 30 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 promoter in GSB-T7 primer <400> 6 taatacgact cactataggg 20 <210> 7 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> GSB-RP primer <400> 7 cctatcccct gtgtgccttg gcagtctcag tcctgcgatc tagatcgg 48

Claims (8)

Preparing a BAC library by extracting genomic DNA to be analyzed and collecting each BAC clone in a 384-well plate to prepare a cell stock;
Three-dimensional BAC clones of the 384-well plate for cell stock are three-dimensionally combined to prepare a three-dimensional BAC library consisting of 60 stores;
Amplifying the BAC clones of the 3-dimensional BAC library using an Escherichia coli culture and extracting BAC clone DNA;
Cutting the extracted BAC clone DNA to a size suitable for analysis;
Attaching a Y-type adapter primer to both ends of the fragment after smoothing both ends of the DNA fragment;
Amplifying both terminal sequences of the genomic DNA using primers comprising the SP6 promoter sequence of the BAC vector or a primer comprising the T7 promoter sequence;
Purifying the amplified DNA and fractionating it into a size suitable for NGS sequence analysis;
Amplifying the fractionated DNA by emPCR;
Analyzing the sequence of the amplified DNA with NGS to obtain sequence data; And
And identifying the location of the BAC clone using the sequence data. &Lt; Desc / Clms Page number 19 &gt; The method according to claim 1,
To label the 384-well plate for cell stocks with marker numbers in rows, columns and plates so as to distinguish the origins of the collected genomic DNA on the X axis (horizontal), Y axis (vertical) and Z axis Lt; / RTI &gt; 3. The method of claim 2,
The three-dimensional BAC library comprises:
Collecting 24 BAC clones on the horizontal axis (X axis) corresponding to the same number in 384-well plates for cell stock in one reservoir;
Collecting 16 BAC clones in the vertical axis (Y axis) corresponding to the same number in a 384-well plate for cell stock into one reservoir; And
(Z axis) by collecting 384 BAC clones contained in one 384-well plate for cell stock into one reservoir. The method according to claim 1,
Wherein the Y-type adapter primer has the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and the terminal nucleotide sequence of the five nucleotide sequences is an unconformable primer. The method according to claim 1,
Wherein the extracted BAC clone DNA is cut using ultrasonic waves. The method according to claim 1,
Wherein the primer comprising the SP6 promoter sequence is a GSB-SP6 primer consisting of the forward adapter of SEQ ID NO: 3, the barcode sequence representing the position in the 3D BAC library, and the SP6 promoter of SEQ ID NO: 4. The method according to claim 1,
The primer comprising the T7 promoter sequence comprises a forward adapter of SEQ ID NO: 5, a barcode sequence representing the position in the 3D BAC library, and GSB-T7 comprising the T7 promoter of SEQ ID NO: 6 Primer. &Lt; / RTI &gt; The method according to claim 1,
The amplification of both end sequences of the genomic DNA is performed using the GSB-SP6 primer of SEQ ID NO: 6, the GSB-RP primer of SEQ ID NO: 7, or the GSB-T7 primer of SEQ ID NO: 7 and the GSB-RP primer of SEQ ID NO: How to.

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