CN110952147B - Method for constructing DNA library for single cell genome sequencing - Google Patents

Abstract

The invention relates to a method for constructing a DNA library for single-cell genome sequencing, which comprises the following steps: fragmenting DNA in the cell nucleus to obtain a fragmented cell nucleus of the DNA; the method comprises the steps of carrying out multiple rounds of marking on fragmented DNA in a plurality of cell nuclei by adopting different sequence tags, so that the fragmented DNA in each cell nucleus is connected with a tag code consisting of the plurality of sequence tags, and the tag codes connected with the fragmented DNA of each cell nucleus are different; and amplifying the fragmented DNA connected with the tag code to obtain a DNA library for sequencing a single cell genome. The method is quick and has low cost.

Description

Method for constructing DNA library for single cell genome sequencing

Technical Field

The invention relates to biotechnology, in particular to a method for constructing a DNA library for single-cell genome sequencing.

Background

Single cell genome sequencing technology is a technology that amplifies and sequences the whole genome at the single cell level. The principle is that the whole genome DNA of the isolated single cell is amplified to obtain a complete genome with high coverage rate, and then high-throughput sequencing is carried out, so that the method can be used for revealing individual differences and cell evolutionary relations in cell populations.

Currently, single cell genomic sequencing requires first the construction of a single cell genomic library and then sequencing analysis. However, when constructing a single cell genomic library, the single cells are separated by relying on an expensive microfluidic platform and reagents, and the operation is complicated and the cost is high.

Disclosure of Invention

Based on this, it is necessary to provide a rapid and low-cost method for constructing a DNA library for single-cell genome sequencing.

A method of constructing a DNA library for single cell genomic sequencing, comprising:

fragmenting DNA in the cell nucleus to obtain a fragmented cell nucleus of the DNA;

carrying out multiple rounds of marking on the fragmented DNA in the cell nuclei by adopting different sequence tags, so that the fragmented DNA in each cell nucleus is connected with a tag code consisting of the sequence tags, and the tag codes connected with the fragmented DNA in each cell nucleus are different; a kind of electronic device with high-pressure air-conditioning system

Amplifying the fragmented DNA connected with the tag code to obtain a DNA library for sequencing a single cell genome.

The method for constructing the DNA library for sequencing the genome of the cells. The cell nuclei are used as reaction chambers for marking DNA, different sequence labels are adopted to carry out multi-round marking on the DNA in the cell nuclei, and finally, the DNA in each cell nucleus is connected with a unique label code formed by multi-round marking, and different cell nuclei are distinguished by the label code, so that the distinction of single cells is realized. The method is simple and convenient to operate and low in cost.

Drawings

FIG. 1 is a single-cell DNA fragment distribution diagram of example 1; FIG. 2 is a graph showing the cell differentiation efficiency of example 1.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Some embodiments of the invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

According to the construction method of the DNA library for sequencing the single cell genome, the cell nuclei are used as reaction sites for marking DNA, different sequence labels are adopted to carry out multiple rounds of marking on the DNA in the cell nuclei, and finally, the DNA in each cell nucleus is connected with a unique label code formed by multiple rounds of marking, and different cell nuclei are distinguished by the label code, so that the distinction of single cells is realized. Taking 96 kinds of sequence tags as an example, 96 kinds of tags are formed after one round of tags, 9216 kinds of tags can be formed after two rounds of tags, and 884736 kinds of tags can be formed after three rounds of tags. Therefore, if the number of cells to be distinguished is smaller than 884736, the label codes of DNA ligation in each cell nucleus can be different through three rounds of labeling. The method can realize single cell distinction through multiple rounds of DNA markers (DNA ligation reaction) without using a microfluidic technology to distinguish single cells, is simple and convenient to operate and low in cost, and is a rapid and low-cost construction method of a DNA library for single cell genome sequencing.

Specifically, the method for constructing a DNA library for single cell genome sequencing comprises steps S110 to S130.

Step S110: fragmenting the DNA in the nucleus to obtain a fragmented DNA nucleus.

Specifically, cells are collected and counted, and then cell membranes of the cells are lysed to obtain nuclei; then, DNA in the nucleus is fragmented to obtain a nucleus with fragmented DNA. Fragmenting DNA within the nucleus facilitates sequence tagging.

In this embodiment, the counting of cells is performed by formaldehyde fixation followed by counting. Specifically, the collected cells are first mixed with formaldehyde so that the cells are fixed for counting, and then the fixed cells are counted. Of course, in other embodiments, other cell counting methods commonly used in the art may be used for cell counting.

In this embodiment, the method of fragmenting the DNA in the nucleus is cleavage. Specifically, the enzyme used for cleavage is Dpn II. The DNA within the nucleus is fragmented prior to labelling, such that a sticky end of the DNA appears, facilitating the attachment of the sequence tag to the DNA.

Step S120: the method comprises the steps of carrying out multiple rounds of marking on fragmented DNA in a plurality of cell nuclei by adopting different sequence tags, so that the fragmented DNA in each cell nucleus is connected with a tag code consisting of the plurality of sequence tags, and the tag codes connected with the fragmented DNA of each cell nucleus are different.

Specifically, the sequence tag is a base sequence for forming a tag code. In sequencing, the tag codes of the fragmented DNA junctions in the same cell nucleus are the same, and the tag codes of the fragmented DNA junctions in different cell nuclei are different. Single cell genomic sequencing is achieved by identifying tag codes to distinguish fragmented DNA within different nuclei. The sequence tag includes a recognition portion (barcode) that serves as an identification. In this embodiment, the number of sequence tags is 200, and the base sequences of the recognition portions of the 200 sequence tags are shown in SEQ ID Nos. 1 to 200. Further, the sequence tags further include a connecting portion (linker) for connecting between the sequence tags. Further, the recognition portion and the linking portion are linked by base-pairing.

Of course, in other embodiments, the tag sequence is not limited to 200, and may be selected according to the total number of cells to be discriminated and the number of rounds of labeling, for example, 48, 96, 384, or the like. The base sequence of the recognition portion of the sequence tag is not limited to the base sequences shown in SEQ ID Nos. 1 to 200, and may be selected according to actual requirements as long as the recognition function thereof is enabled.

Further, the fragmented DNA in the plurality of nuclei is labeled with different sequence tags for a plurality of rounds, so that the tag codes composed of the plurality of sequence tags are connected to the fragmented DNA in each nucleus, and the steps of the tag codes connected to the fragmented DNA in each nucleus are different, including the steps S121 to S123.

Step S121: after grouping the cell nuclei with the fragmented DNA, marking the fragmented DNA in each group of cell nuclei by adopting different first sequence labels, so that the fragmented DNA in each group of cell nuclei is connected with the first sequence labels, and the first sequence labels connected with the fragmented DNA of each group of cell nuclei are different, thereby obtaining a plurality of groups of primary marked cell nuclei. Wherein the first sequence tag comprises a first sequence for identification and a first linking sequence for linking to the second sequence tag. The first sequence is connected with the first connecting sequence, and the first connecting sequence and the second sequence tag are connected in a base complementary pairing mode. Further, the 5' end of the first sequence is linked to a phosphate group. The first sequence can be linked to the fragmented DNA by a phosphate group.

In this embodiment, the base sequence of the first sequence is selected from one of the base sequences shown in SEQ ID No.1 to SEQ ID No. 96; the base sequence of the first connecting sequence is shown as SEQ ID No. 201. Of course, the base sequence of the first sequence is not limited to one of the base sequences shown in SEQ ID Nos. 1 to 96. In other embodiments, other base sequences commonly used in the art for labeling or base sequences designed according to methods conventional in the art for labeling; similarly, the base sequence of the first ligation sequence is not limited to the base sequence shown in SEQ ID No. 201.

In the present embodiment, the cell nuclei of the plurality of cells are grouped in a random uniform manner.

Specifically, grouping a plurality of cell nuclei with fragmented DNA, mixing the cell nuclei with different first sequence tags, and incubating the cell nuclei to obtain a plurality of groups of pre-ligation liquid containing the different first sequence tags; and adding DNA ligase into each group of pre-ligation liquid, and then incubating to obtain a plurality of groups of primary marked cell nuclei. After the first sequence tag is mixed and incubated with the cell nucleus, the ligase is added for incubation, so that the first sequence tag enters the cell nucleus and is mixed with the fragmented DNA in the cell nucleus, a plurality of fragmented DNA are separated by the first sequence tag, and interconnection among the plurality of fragmented DNA is reduced.

Further, the plurality of DNA fragmented nuclei are aliquoted into different reaction vessels (e.g., EP tubes or multiwell plates) containing the first sequence tags, wherein the first sequence tags in the different reaction vessels are different; then incubating to obtain a plurality of groups of pre-ligation solutions containing different first sequence tags. And adding DNA ligase into the plurality of groups of pre-ligation liquid containing different first sequence tags and incubating to enable the fragmented DNA in the cell nucleus in the reaction container to perform ligation reaction with the first sequence tags, thereby obtaining a plurality of groups of primary marked cell nuclei. Wherein, the fragmented DNA in the cell nucleus in the same reaction vessel is connected with the same first sequence label, and the first sequence labels connected with the fragmented DNA in the cell nucleus in different reaction vessels are different.

In one embodiment, after grouping the plurality of DNA fragments into the nuclei, the step of labeling the fragmented DNA in each of the plurality of nuclei with different first sequence tags, so that the fragmented DNA in each of the plurality of nuclei is connected to the first sequence tags, and the first sequence tags connected to the fragmented DNA in each of the plurality of nuclei are different, and the step of mixing each of the plurality of primary labeled nuclei with the blocking sequence, is further included. Typically, an excess of the first sequence tag is used in combination with the nucleus such that all fragmented DNA within the nucleus is ligated to the first sequence tag, so that after the ligation reaction is completed, there is a free first sequence tag. At this time, if the nuclei in the respective reaction vessels are directly mixed, the labeling of the next round may be disturbed. Thus, by adding a blocking sequence to the reaction vessel after the ligation reaction has ended, the blocking sequence binds to the free first sequence tag in each reaction vessel, reducing the effect of the previous round of labelling on the next round of labelling.

In this embodiment, the base sequence of the blocking sequence is shown in SEQ ID No. 203. Of course, the base sequence of the blocking sequence is not limited to the base sequence shown in SEQ ID No. 203. In other embodiments, other base sequences commonly used in the art for blocking or base sequences designed to act as blocking according to methods conventional in the art are also possible.

Step S122: and mixing and grouping a plurality of groups of primary marked cell nuclei, and then marking the segmented DNA in the grouped primary marked cell nuclei by adopting different second sequence tags, so that the segmented DNA in each group of primary marked cell nuclei is connected with the second sequence tags, and the second sequence tags connected with the segmented DNA of each group of primary marked cell nuclei are different, thereby obtaining a plurality of groups of secondary marked cell nuclei. Wherein the second sequence tag comprises a second sequence for identification and a second linking sequence for linking to the first sequence tag.

In this embodiment, the base sequence of the second sequence is selected from one of the base sequences shown as SEQ ID No.97 to SEQ ID No. 192; the base sequence of the second connecting sequence is shown as SEQ ID No. 202. Of course, in other embodiments, the base sequence of the second sequence is not limited to one of the base sequences shown in SEQ ID No.97 to SEQ ID No.192 described above, but may be other base sequences commonly used in the art or base sequences designed for labeling according to a conventional method in the art; similarly, the base sequence of the second junction sequence is not limited to the base sequence shown in SEQ ID No. 202.

Further, biotin is attached to the 5' end of the second sequence to facilitate subsequent purification of the fragmented DNA with tag code attached thereto.

In this embodiment, the mode of grouping the plurality of sets of primary marker nuclei after mixing is a random equal grouping.

More specifically, a plurality of primary marker nuclei are equally divided and mixed in different reaction vessels containing the second sequence tags, wherein the second sequence tags in the different reaction vessels are different; then incubating to obtain a plurality of groups of pre-ligation solutions containing different second sequence tags. And adding DNA ligase into the plurality of groups of pre-ligation liquid containing different second sequence tags and incubating to enable the first sequence tags and the second sequence tags of the fragmented DNA in the cell nuclei in the reaction container to perform ligation reaction, thereby obtaining a plurality of groups of secondary marked cell nuclei. Wherein, the fragmented DNA in the cell nucleus in the same reaction vessel is connected with the same second sequence label, and the second sequence labels connected with the fragmented DNA in the cell nucleus in different reaction vessels are different.

Step S123: and mixing and grouping a plurality of groups of secondary marked cell nuclei, and then marking the fragmented DNA in the grouped secondary marked cell nuclei by adopting different third sequence tags, so that the fragmented DNA of each group of secondary marked cell nuclei is connected with the third sequence tags, and the third sequence tags connected with the DNA of each group of secondary marked cell nuclei are different from each other, thereby obtaining a plurality of groups of tertiary marked cell nuclei. Wherein the third sequence tag comprises a third sequence for identification.

In this embodiment, the base sequence of the third sequence is selected from one of the base sequences shown in SEQ ID Nos. 193 to 200. Of course, in other embodiments, the base sequence of the third sequence is not limited to one of the base sequences shown in SEQ ID Nos. 193 to 200 described above, but may be other base sequences commonly used in the art or base sequences designed for labeling according to a conventional method in the art.

More specifically, the plurality of secondary marker nuclei are equally divided and mixed in different reaction vessels containing the third sequence tag, wherein the third sequence tag is different in the different reaction vessels; then incubating to obtain a plurality of groups of pre-ligation solutions containing different third sequence tags. And adding DNA ligase into the plurality of groups of pre-ligation liquid containing different third sequence tags and incubating to enable the second sequence tags and the third sequence tags of the fragmented DNA in the cell nuclei in the reaction vessel to generate ligation reaction, thereby obtaining a plurality of groups of tertiary marked cell nuclei. Wherein, the fragmented DNA in the cell nucleus in the same reaction vessel is connected with the same third sequence label, and the third sequence labels connected with the fragmented DNA in the cell nucleus in different reaction vessels are different.

In the present embodiment, the first sequence tag, the second sequence tag, and the third sequence tag each have a different sequence that functions as a marker. Of course, in other embodiments, the sequences of the first, second, and third sequence tags that serve as identifiers may be identical. For example, the first sequence of the first tag and the second sequence of the second tag are each the base sequences shown in SEQ ID No.1 to SEQ ID No. 96.

In this embodiment, the tag code of the DNA in each nucleus is formed by three rounds of labeling. The product of the number of types of the first sequence tag and the number of types of the second sequence tag and the number of types of the third sequence tag is greater than the number of nuclei into which the DNA is fragmented; after three rounds of labeling, the tag code of the DNA in each cell nucleus is formed by sequentially connecting a first sequence tag, a second sequence tag and a third sequence tag corresponding to the DNA in each cell nucleus. Of course, the number of rounds required for forming the tag codes for DNA for discriminating between different nuclei is not limited to three, and may be designed according to the number of nuclei and the number of kinds of sequence tags to be discriminated.

Of course, after each different step of obtaining the tag code linked to the fragmented DNA of each of the nuclei, a step of lysing the nuclei and purifying the DNA linked to the tag code is further included. For example, if the formation of the tag code requires only three rounds, the step of obtaining the tertiary-labeled nuclei may further include lysing the tertiary-labeled nuclei and purifying the released DNA linked to the first, second and third sequence tags to obtain DNA linked to the tag code.

Step S130: and amplifying the fragmented DNA connected with the tag codes to obtain a DNA library for sequencing a single cell genome.

Specifically, fragmenting the fragmented DNA connected with the tag code by adopting a fragment technology, and connecting the fragmented DNA with the tag code to a database building joint to obtain a plurality of fragmented DNA with shorter length and connected with the tag code; then, the fragmented DNA with the tag code attached thereto having a shorter length is amplified to obtain a DNA library for single cell genome sequencing. Of course, in other embodiments, other methods commonly used in the art may be used to fragment the fragmented DNA with the attached tag code and attach a library linker.

In another embodiment, the method for constructing a DNA library for single-cell genomic sequencing, which includes the steps of making the tag codes for the ligation of fragmented DNA of each cell nucleus different from each other, includes the steps of:

after grouping a plurality of cell nuclei with fragmented DNA, marking the fragmented DNA in each group of cell nuclei by adopting different first sequence tags, so that the fragmented DNA in each group of cell nuclei is connected with the first sequence tags, the first sequence tags connected with the fragmented DNA of each group of cell nuclei are different, and a plurality of groups of primary marked cell nuclei are obtained, wherein the first sequence tags are sequence tags;

mixing and grouping a plurality of groups of primary marked cell nuclei, and then marking the segmented DNA in the grouped primary marked cell nuclei by adopting different second sequence tags, so that the segmented DNA in each group of primary marked cell nuclei is connected with the second sequence tags, the second sequence tags connected with the segmented DNA of each group of primary marked cell nuclei are different, and a plurality of groups of secondary marked cell nuclei are obtained, wherein the second sequence tags are sequence tags;

uniformly grouping a plurality of groups of secondary marked cell nuclei after mixing, wherein the number of the cell nuclei of each group is smaller than the product of the number of the types of the first sequence tags and the number of the types of the second sequence tags, so as to obtain a plurality of groups of to-be-lysed liquid, and tag codes consisting of the first sequence tags and the second sequence tags are connected to fragmented DNA in each cell nucleus in the to-be-lysed liquid, and the tag codes connected with the fragmented DNA of each cell nucleus are different; or the probability of the same label code of the fragmented DNA connection of the cell nuclei in the same group of to-be-lysed liquid is less than 5%;

and (3) splitting one group of to-be-split solution to release fragmented DNA (deoxyribonucleic acid) connected with a first sequence tag and a second sequence tag in each cell nucleus in the to-be-split solution, then fragmenting the fragmented DNA connected with the first sequence tag and the second sequence tag by a fragment technology, and connecting a library building joint containing a third sequence tag to obtain a plurality of fragmented DNA connected with tag codes consisting of the first sequence tag, the second sequence tag and the third sequence tag, wherein the tag codes of the fragmented DNA connected with the third sequence tag are different.

In the method for constructing a DNA library for sequencing a single-cell genome according to this embodiment, a plurality of sets of secondary marker nuclei are mixed and equally grouped, and the number of nuclei in each set is smaller than the product of the number of the first sequence tags and the number of the second sequence tags, instead of the third marker, after each set of nuclei is lysed, a tag code composed of the first sequence tags and the second sequence tags is linked to fragmented DNA in each nucleus, and the tag codes linked to the fragmented DNA in each nucleus are different.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following is a detailed description of specific embodiments. The drugs and apparatus used in the examples are all routine choices in the art, unless specifically indicated. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.

Example 1

(1) The Shanghai Biotechnology services Limited were commissioned to synthesize the first sequence tag, the second sequence tag, the blocking sequence and the library linker. Wherein: the first sequence tags are 96, each first sequence tag consists of a first connecting sequence and a first sequence connected with the first connecting sequence, the first sequence has 96 types, the base sequences of the 96 types of first sequences are shown as SEQ ID No. 1-SEQ ID No.96, the 5' ends of the 96 types of first sequences are connected with phosphate groups, and the base sequences of the first connecting sequences of the 96 types of first sequence tags are shown as SEQ ID No. 201. 96 kinds of second sequence labels are provided, each second sequence label consists of a second connecting sequence and a second sequence connected with the second connecting sequence, the second sequence has 96 kinds, the base sequences of the 96 kinds of second sequences are shown as SEQ ID No. 97-SEQ ID No.192, the 5' ends of the 96 kinds of second sequences are connected with biotin, and the base sequences of the second connecting sequences of the 96 kinds of second sequence labels are shown as SEQ ID No. 202. The blocking sequence can be complementarily paired with the base at the 5' end of 96 first connecting sequences, and the base sequence of the blocking sequence is shown as SEQ ID No. 203. The library-building joint comprises an i5 terminal joint and 8 i7 terminal joints, the base sequence of the i5 terminal joint is shown as SEQ ID No.204, the base sequences of the 8 i7 terminal joints are shown as SEQ ID No. 205-SEQ ID No.212, the 8 i7 terminal joints comprise 8 third sequence tags, and the sequences of the 8 third sequence tags are shown as 8 SEQ ID No. 193-SEQ ID No. 200.

(2) Cells were collected and crosslinked: human cells (293T) and murine cells (CT 26) were collected and cross-linked, respectively, and the procedure for both human and murine cells was as follows: A. centrifugation to collect freshly cultured cells 1X 10 ⁶ And 1500rpm,3min, and resuspended in 1mL DMEM medium. B. Add 312.5mu.L of 16% formaldehyde (1% concentration) was fixed in the cell suspension of step A and incubated at room temperature for 10min with spin. C. To the cell suspension incubated in step B, 312.5. Mu.L of 2M glycine (final concentration of 0.125M) was added, and the incubation was rotated at room temperature for 5min, to terminate the crosslinking reaction. Then incubated on ice for 15min. D. Cells were collected by centrifugation at 1500rpm for 3 min. E. The 1 XPBS buffer was washed once. F. After discarding the supernatant, the cells may be directly lysed to extract nuclei, or they may be temporarily stored at-80 ℃.

(3) Lyse cells and fragment DNA within the nucleus:

A. separately calculating the human cells and the mouse cells obtained in the step (2), and then according to the following steps of 1:1 to give a total number of cells of 1X 10 ⁵ And each. B. Add 500. Mu.L of pre-chilled lysis buffer (mixture of 10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA-630, EDTA-free protease inhibitor) to the mixture of human and murine cells obtained in step A, fully resuspend, incubate on ice for 30min, and allow the cells to lyse fully. C. The supernatant was removed by centrifugation at 650g for 5min at 4℃and the nuclei were collected. D. The nuclei were washed twice with 500. Mu.L of 1 XDpn II buffer. E. 362 u L1 x Dpn II heavy suspension cell nuclei. F. Increase nuclear membrane permeability: add 38. Mu.L of 1% SDS to the nuclei of step E and carefully blow mix to avoid air bubbles. After incubation for 10min at 65℃the ice was quickly inserted and 44. Mu.L 10% Triton X-100 was added and carefully blown to mix to avoid air bubbles. G. Digestion of chromosomes: after increasing nuclear membrane permeability, 50. Mu.L of 1% BSA, 10. Mu.L of 10 XDpn II buffer and 20. Mu.L of Dpn II (NEB) were added and incubated at 37℃overnight with spin (50 rpm).

(4) Labeling of DNA in the nucleus

A. The nuclei were treated at 65℃for 20min to inactivate Dpn II. B. The nuclei were sequentially passed through filters having pore diameters of 40 μm and 20 μm to remove the adhered cell clusters. C. 2 96-well plates were prepared, and first and second sequence tags were prepared: 1) First sequence tag: of the 96 first sequence tags used in the first round, each first sequence had a final concentration of 14. Mu.M and the first ligation sequence had a final concentration of 13. Mu.M. Firstly, adding 14 mu L of first sequence into each reaction well of a 96-well plate, wherein the first sequences in the wells are different; then 13. Mu.L of the first ligation sequence was added to each of the reaction wells to which the first sequence was added; finally 73. Mu.L of water was added to each reaction well to which the first ligation sequence was added. 2) Second sequence tag: of the 96 second sequence tags used in the second round, each second sequence had a final concentration of 16. Mu.M and the second ligation sequence had a final concentration of 15. Mu.M. Firstly, adding 16 mu L of second sequences into each reaction well of a 96-well plate, wherein the second sequences in the reaction wells are different; then 15. Mu.L of the second ligation sequence was added to each of the wells to which the second sequence was added; finally 69. Mu.L of water was added to each reaction well to which the second ligation sequence was added. Before use, for each 96-well plate, annealing was performed with the following thermal cycling operation: heating to 95deg.C for 2min; then the temperature is reduced to 20 ℃ with the speed of-0.1 ℃/s; then, at 4℃a 96-well plate for the first round of labeling and a 96-well plate for the second round of labeling were obtained. D. The first round of connection: 1) The nuclear solution was prepared in accordance with Table 1, and then the obtained nuclear solution was dispensed into each of the reaction wells of the 96-well plate for the first round of labeling, 10. Mu.L of each reaction well, and was thoroughly blown and mixed with a gun head. Then, the mixture was covered with a plywood seal, and incubated in an incubator at 37℃for 30 minutes with slow rotation.

TABLE 1

2) The ligase solution was prepared in accordance with Table 2, and the ligase solution was dispensed into reaction wells of a 96-well plate to which the cell nuclei and the ligase buffer had been added, 10. Mu.L of each reaction well was then mixed by blowing with a gun head. Then covered with a plywood seal and incubated at room temperature for 2 hours with slow rotation.

TABLE 2

3) Blocking of the first round connection: 10. Mu.L of blocking sequence was added to each well after the incubation of step 2), sealed with self-adhesive sealing plate membrane and incubated for 30min at 37℃in incubator with slow rotation (50 rpm).

E. After blocking, the 96-well plate is taken out, the sealing plate film is taken down, and all cell nuclei are transferred into a liquid separating tank for merging. F. After passing through a 20 μm filter, the mixture was transferred to a fresh liquid tank to remove the adhered cell nucleus. G. The second wheel is connected with: 100 mu L T DNA ligase was added to the nuclear solution and mixed by blowing 20 times to avoid air bubbles. The nuclei were transferred to 96-well plates with annealed second round of labeling, 28 μl per reaction well. Incubation was carried out in a 37℃incubator with slow rotation (50 rpm) for 30min. H. Terminating the ligation reaction: the termination reaction solution (composed of 400mL 0.5M EDTApH8.0 and 800mL H) was added to a new liquid separation tank ₂ O composition). And C, transferring the cell nucleus after the incubation in the step G into a liquid separating tank, fully blowing and uniformly mixing the cell nucleus and the termination reaction liquid during each transfer, and then adding new cell nucleus. I. All nuclei were transferred to a 15mL centrifuge tube to give about 5mL of a secondary labeled nuclear solution, and the fragmented DNA within the secondary labeled nuclei was sequentially linked with a first sequence tag and a second sequence tag.

(5) DNA and proteolytic crosslinking, lysing nuclei:

A. 2 Xlysis buffer was prepared as in Table 3:

TABLE 3 Table 3

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B. The following wash buffers were prepared:

TABLE 4 Table 4

Reagent(s)	Volume of
		1×PBS	4000μL
10％Triton X-100	40μL

C. According to the weight of 100:1 to the secondary labeled nuclei solution obtained in step (4) (Triton X-100 final concentration 0.1%). D. After centrifugation at 1000g at 4℃for 5min and careful removal of the supernatant, the pellet was resuspended in 4mL of wash buffer and thoroughly mixed to wash the nuclei. E. Centrifuge at 1000g for 5min at 4 ℃. The supernatant was then aspirated and resuspended in 50 μl PBS. F. mu.L to 5. Mu.L of 1 XPBS were taken and counted with platelets. H. The number of cells contained in the sub-libraries is determined according to the types of the first sequence tag and the second sequence tag, and in this embodiment, the number of cells in each sub-library is less than 1800. I. The number of cells required for each sub-pool was placed in a new 1.7mL tube. 1 XPBS was added to each tube and the final volume was 50. Mu.L. J. mu.L of 2 Xlysis buffer was added to each tube. K. To each lysate was added 10. Mu.L proteinase K (20 mg/mL). L, at 55 ℃ for 2 hours or overnight.

(6) Purification of fragmented DNA with first and second sequence tags attached:

A. mu.L of streptavidin beads were added to a 1.5 ml tube containing 400. Mu. L Tween Wash Buffer (TWB). Spin mixing at room temperature for 2 min. Wherein the TWB formulation is as shown in Table 5:

TABLE 5

Liquid storage	Final concentration	10mL	50mL
				1M Tris-HCl pH 8.0	5mM	50μL	250μL
0.5M EDTApH 8.0	0.5mM	10μL	50μL
				5M NaCl	1M	2mL	10mL

B. The centrifuge tube is placed on a magnetic rack, and the supernatant is sucked off until the solution becomes clear. C. The steps a and B are repeated again. D. With dd H ₂ O increases the volume of the nuclear lysate obtained in step C to 400. Mu.L. E. The beads were resuspended in 400. Mu.L of 2 Xbinding buffer (BB) and 400. Mu.L of nuclear lysis solution. The formulation of the 2×binding buffer (BB) is shown in table 6:

TABLE 6

Liquid storage	Final concentration	10mL	50mL
				1M Tris-HCl pH 8.0	10mM	100μL	500μL
0.5M EDTApH 8.0	1mM	20μL	100μL
				5M NaCl	2M	4mL	20mL

F. The biotin-labeled fragments were bound to streptavidin magnetic beads by spin incubation at room temperature for 15min. G. The centrifuge tube is placed on a magnetic rack. After the solution became clear, the supernatant was discarded. H. The beads were resuspended with 400. Mu.L of 1 Xbinding buffer and transferred to a new LoBind tube. I. The centrifuge tube is placed on a magnetic rack. After the solution became clear, the supernatant was discarded. J. The beads were resuspended with 100. Mu.L of 1 Xbinding buffer and transferred to a new LoBind tube. K. The centrifuge tube is placed on a magnetic rack. After the solution became clear, the supernatant was discarded. I. Add 20. Mu.L ddH ₂ O resuspended magnetic beads.

(7) The DNA was fragmented and inserted into a library linker using the fragment technique:

A5×TTBL buffer was thawed on ice and the tab reaction was performed as in Table 7: TTBL, purified magnetic bead-DNA, TTE Mix V5 and H are firstly carried out ₂ Mixing the mixed solution of O thoroughly to avoid foaming, incubating at 55deg.C for 10min, and rapidly cooling to 4deg.C. Finally 7.5. Mu.L of 1% SDS was added to the tube and mixed well with blowing, and incubated at 55℃for 15min.

TABLE 7

B. The centrifuge tube is placed on a magnetic rack. After the solution became clear, the supernatant was discarded. C. The beads were resuspended at 800. Mu.L 1 XBB and transferred to a new LoBind tube. D. The centrifuge tube is placed on a magnetic rack. After the solution became clear, the supernatant was discarded. E. The beads were resuspended with 100. Mu.L of 1 XBB and transferred to a new LoBind tube. The centrifuge tube is placed on a magnetic rack, and the supernatant is removed after the solution becomes clear. F. 20 mu L ddH ₂ O resuspended magnetic beads.

(8) Library amplification: kit V2, TD502 was prepared using the Vazyme TruePrepTM DNA library from Vazyme company. Wherein the amplification system is shown in Table 8, and the amplification conditions are shown in Table 9.

TABLE 8

PCR Mix	50μL
		5×TAB	10μL
TAE	1μL
		i5 end cap (2.5 mu M)	1μL
i7 end cap (2.5 mu M)	1μL
		H 2 O	17μL
The magnetic beads obtained in the step (6)	20μL

TABLE 9

(9) Library amplification: the fragment was subjected to fragment sorting and purification using AMpure XP magnetic beads for removing primer dimer and obtaining DNA fragments of 300bp to 500 bp:

A. vazyme VAHTS DNA beads were allowed to stand at room temperature for 30min before use and equilibrated to room temperature. B. The supernatant of the PCR product was gently centrifuged. And DNA magnetic beads were added to the supernatant of the PCR product in a ratio of 0.55X. C. Repeatedly blowing for at least 10 times, and fully and uniformly mixing. D. Standing at room temperature for 5min. E. The beads were attached with a magnetic rack for about 5min, and then the supernatant was transferred to a new tube. F. To the supernatant was added 0.15 x volume of magnetic beads. Repeatedly blowing for at least 10 times, and fully and uniformly mixing. G. Standing at room temperature for 5min. H. The beads were attached with a magnetic rack for about 5min. The supernatant was discarded. I. The beads were washed twice with 1mL of freshly prepared 70% ethanol, taking care not to attract the beads. J. After the supernatant was aspirated, the tube was placed on a magnetic rack and the beads were air dried. K. The beads were resuspended in 30. Mu.L ddH ₂ In O, the mixture is blown for more than 10 times to be fully and uniformly mixed. L, standing at room temperature for 10min, and tapping the test tube every 2 min. And M, placing the centrifuge tube on a magnetic rack for standing for 5min. N, the supernatant containing the final library was transferred to a new centrifuge tube. O, as described above, library size was detected using 2% agarose gel (5. Mu.L library) electrophoresis,and quantitated (1. Mu.L library) by Qubit. P, the library is sent to Shenzhen sea pulos biotechnology Co., ltd for on-machine sequencing, the sequencing mode is PE150 sequencing, and the sequencing platform is HiSeq X Ten.

(10) And carrying out quality analysis on the sequencing data by adopting a bioinformatics method.

A. The parameters are default parameters for comparing read1 containing genomic information to human and mouse reference genomes using bwa mem. B. Fragments in read1 can be aligned to the genome and alignment information recorded to confirm that the read is from the species. C. The read2 containing the first, second and UMI (Unique molecular identifiers) sequences identified was quality controlled using fastp, using parameter-a to preserve the linker sequence. D. And extracting the barcode1, the barcode2 and the UMI in the rest read2 file. E. Clustering the extracted sequence tags and UMI by using a starcode, and setting the allowed maximum editing distance to be 1 by using a parameter-d. F. Reads containing sequence tags that are not present in the tag library are removed. G. Reads containing the same sequence tag combinations are grouped into the same cluster, while the reads are de-duplicated according to UMI information, and then the number of reads of human and murine origin contained in each cluster is noted based on species information extracted by read 1. H. Histograms of the numbers of human source and mouse source reads of each group are respectively drawn, the histograms are generally in bimodal distribution, and sites which can be just separated from two peaks are selected as threshold values. Each cluster is then categorized as follows: a) If the numbers of the human source reads and the mouse source reads contained in a certain group are lower than the corresponding threshold values, classifying the group as non-cell;

b) If the number of human reads contained in a group is higher than the corresponding threshold value, and more than 90% of reads in the group are human, the group is classified as human cells.

c) If the number of the reads of the mouse sources contained in a certain group is higher than the corresponding threshold value, and more than 90% of reads of the group are the mouse sources, the mouse sources are classified as mouse cells.

d) If the conditions in a), b), c) are not met, they are classified as "mixed cells".

After the machine-down data are processed according to the step (10), a single-cell DNA fragment number distribution diagram (shown in figure 1) and a human and mouse cell distinguishing efficiency diagram (shown in figure 2) can be obtained. In FIG. 1, the abscissa indicates the distribution of the number of non-redundant genomic DNA fragments obtained in a single cell, and the ordinate indicates the number of cells. In fig. 2, the light grey (in the upper left part of the figure) indicates single cells of mouse origin that successfully underwent single cell labelling, each cell containing only one tag code. Light black (in the lower right part of the figure) indicates successfully labeled cells of human origin, each cell containing only one tag code. Black (in the upper right part of the figure) indicates that one sequence tag marks DNA of both mouse and human origin, i.e. the sequence tag code is contaminated, and single cells cannot be distinguished, which part has a cell proportion of 4.62% and is within an acceptable range of single cell contamination (< 5%). Dark grey (in the lower left part of the figure) is background noise or a DNA fragment that failed to be labeled. The abscissa and the overall coordinate of fig. 2 each represent the number of reads contained in each single cell.

Thus, as can be seen from FIGS. 1 and 2, the method of example 1 can be used to distinguish single cells and can be used to construct DNA libraries for single cell genomic sequencing.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description. The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> university of south science and technology

<120> method for constructing DNA library for single cell genome sequencing

<160> 212

<170> SIPOSequenceListing 1.0

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catcggcgta cgactaccac tgtatccacg tgcttgag 38

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catcggcgta cgactacatt ggcatccacg tgcttgag 38

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catcggcgta cgactcagat ctgatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

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catcggcgta cgactcatca agtatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

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catcggcgta cgactcgctg atcatccacg tgcttgag 38

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<211> 38

<212> DNA

<213> Artificial Sequence

<400> 10

catcggcgta cgactacaag ctaatccacg tgcttgag 38

<210> 11

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 11

catcggcgta cgactctgta gccatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

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catcggcgta cgactagtac aagatccacg tgcttgag 38

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<213> Artificial Sequence

<400> 13

catcggcgta cgactaacaa ccaatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

<400> 14

catcggcgta cgactaaccg agaatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

<400> 15

catcggcgta cgactaacgc ttaatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

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catcggcgta cgactaagac ggaatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

<400> 17

catcggcgta cgactaaggt acaatccacg tgcttgag 38

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<212> DNA

<213> Artificial Sequence

<400> 18

catcggcgta cgactacaca gaaatccacg tgcttgag 38

<210> 19

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 19

catcggcgta cgactacagc agaatccacg tgcttgag 38

<210> 20

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 20

catcggcgta cgactacctc caaatccacg tgcttgag 38

<210> 21

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 21

catcggcgta cgactacgct cgaatccacg tgcttgag 38

<210> 22

<211> 38

<212> DNA

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<400> 22

catcggcgta cgactacgta tcaatccacg tgcttgag 38

<210> 23

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 23

catcggcgta cgactactat gcaatccacg tgcttgag 38

<210> 24

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 24

catcggcgta cgactagagt caaatccacg tgcttgag 38

<210> 25

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 25

catcggcgta cgactagatc gcaatccacg tgcttgag 38

<210> 26

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 26

catcggcgta cgactagcag gaaatccacg tgcttgag 38

<210> 27

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 27

catcggcgta cgactagtca ctaatccacg tgcttgag 38

<210> 28

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 28

catcggcgta cgactatcct gtaatccacg tgcttgag 38

<210> 29

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 29

catcggcgta cgactattga ggaatccacg tgcttgag 38

<210> 30

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 30

catcggcgta cgactcaacc acaatccacg tgcttgag 38

<210> 31

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 31

catcggcgta cgactgacta gtaatccacg tgcttgag 38

<210> 32

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 32

catcggcgta cgactcaatg gaaatccacg tgcttgag 38

<210> 33

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 33

catcggcgta cgactcactt cgaatccacg tgcttgag 38

<210> 34

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 34

catcggcgta cgactcagcg ttaatccacg tgcttgag 38

<210> 35

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<400> 35

catcggcgta cgactcatac caaatccacg tgcttgag 38

<210> 36

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<400> 36

catcggcgta cgactccagt tcaatccacg tgcttgag 38

<210> 37

<211> 38

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<213> Artificial Sequence

<400> 37

catcggcgta cgactccgaa gtaatccacg tgcttgag 38

<210> 38

<211> 38

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<213> Artificial Sequence

<400> 38

catcggcgta cgactccgtg agaatccacg tgcttgag 38

<210> 39

<211> 38

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<400> 39

catcggcgta cgactcctcc tgaatccacg tgcttgag 38

<210> 40

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<400> 40

catcggcgta cgactcgaac ttaatccacg tgcttgag 38

<210> 41

<211> 38

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<213> Artificial Sequence

<400> 41

catcggcgta cgactcgact ggaatccacg tgcttgag 38

<210> 42

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<400> 42

catcggcgta cgactcgcat acaatccacg tgcttgag 38

<210> 43

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<213> Artificial Sequence

<400> 43

catcggcgta cgactctcaa tgaatccacg tgcttgag 38

<210> 44

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<400> 44

catcggcgta cgactctgag ccaatccacg tgcttgag 38

<210> 45

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<213> Artificial Sequence

<400> 45

catcggcgta cgactctggc ataatccacg tgcttgag 38

<210> 46

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 46

catcggcgta cgactgaatc tgaatccacg tgcttgag 38

<210> 47

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 47

catcggcgta cgactcaaga ctaatccacg tgcttgag 38

<210> 48

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 48

catcggcgta cgactgagct gaaatccacg tgcttgag 38

<210> 49

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 49

catcggcgta cgactgatag acaatccacg tgcttgag 38

<210> 50

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 50

catcggcgta cgactgccac ataatccacg tgcttgag 38

<210> 51

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 51

catcggcgta cgactgcgag taaatccacg tgcttgag 38

<210> 52

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 52

catcggcgta cgactgctaa cgaatccacg tgcttgag 38

<210> 53

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 53

catcggcgta cgactgctcg gtaatccacg tgcttgag 38

<210> 54

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 54

catcggcgta cgactggaga acaatccacg tgcttgag 38

<210> 55

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 55

catcggcgta cgactggtgc gaaatccacg tgcttgag 38

<210> 56

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 56

catcggcgta cgactgtacg caaatccacg tgcttgag 38

<210> 57

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 57

catcggcgta cgactgtcgt agaatccacg tgcttgag 38

<210> 58

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 58

catcggcgta cgactgtctg tcaatccacg tgcttgag 38

<210> 59

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 59

catcggcgta cgactgtgtt ctaatccacg tgcttgag 38

<210> 60

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 60

catcggcgta cgacttagga tgaatccacg tgcttgag 38

<210> 61

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 61

catcggcgta cgacttatca gcaatccacg tgcttgag 38

<210> 62

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 62

catcggcgta cgacttccgt ctaatccacg tgcttgag 38

<210> 63

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 63

catcggcgta cgacttcttc acaatccacg tgcttgag 38

<210> 64

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 64

catcggcgta cgacttgaag agaatccacg tgcttgag 38

<210> 65

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 65

catcggcgta cgacttggaa caaatccacg tgcttgag 38

<210> 67

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 67

catcggcgta cgacttggct tcaatccacg tgcttgag 38

<210> 67

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 67

catcggcgta cgacttggtg gtaatccacg tgcttgag 38

<210> 68

<211> 38

<212> DNA

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<400> 68

catcggcgta cgactttcac gcaatccacg tgcttgag 38

<210> 69

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 69

catcggcgta cgactaactc accatccacg tgcttgag 38

<210> 70

<211> 38

<212> DNA

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<400> 70

catcggcgta cgactaagag atcatccacg tgcttgag 38

<210> 71

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 71

catcggcgta cgactaagga cacatccacg tgcttgag 38

<210> 72

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 72

catcggcgta cgactaatcc gtcatccacg tgcttgag 38

<210> 73

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 73

catcggcgta cgactaatgt tgcatccacg tgcttgag 38

<210> 74

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 74

catcggcgta cgactacacg accatccacg tgcttgag 38

<210> 75

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 75

catcggcgta cgactacaga ttcatccacg tgcttgag 38

<210> 76

<211> 38

<212> DNA

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<400> 76

catcggcgta cgactagatg tacatccacg tgcttgag 38

<210> 77

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 77

catcggcgta cgactagcac ctcatccacg tgcttgag 38

<210> 78

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 78

catcggcgta cgactagcca tgcatccacg tgcttgag 38

<210> 79

<211> 38

<212> DNA

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<400> 79

catcggcgta cgactaggct aacatccacg tgcttgag 38

<210> 80

<211> 38

<212> DNA

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<400> 80

catcggcgta cgactatagc gacatccacg tgcttgag 38

<210> 81

<211> 38

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<400> 81

catcggcgta cgactatcat tccatccacg tgcttgag 38

<210> 82

<211> 38

<212> DNA

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<400> 82

catcggcgta cgactattgg ctcatccacg tgcttgag 38

<210> 83

<211> 38

<212> DNA

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<400> 83

catcggcgta cgactcaagg agcatccacg tgcttgag 38

<210> 84

<211> 38

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<400> 84

catcggcgta cgactcacct tacatccacg tgcttgag 38

<210> 85

<211> 38

<212> DNA

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<400> 85

catcggcgta cgactccatc ctcatccacg tgcttgag 38

<210> 86

<211> 38

<212> DNA

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<400> 86

catcggcgta cgactccgac aacatccacg tgcttgag 38

<210> 87

<211> 38

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<400> 87

catcggcgta cgactcctaa tccatccacg tgcttgag 38

<210> 88

<211> 38

<212> DNA

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<400> 88

catcggcgta cgactcctct atcatccacg tgcttgag 38

<210> 89

<211> 38

<212> DNA

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<400> 89

catcggcgta cgactcgaca cacatccacg tgcttgag 38

<210> 90

<211> 38

<212> DNA

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<400> 90

catcggcgta cgactcggat tgcatccacg tgcttgag 38

<210> 91

<211> 38

<212> DNA

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<400> 91

catcggcgta cgactctaag gtcatccacg tgcttgag 38

<210> 92

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<400> 92

catcggcgta cgactgaaca ggcatccacg tgcttgag 38

<210> 93

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<400> 93

catcggcgta cgactgacag tgcatccacg tgcttgag 38

<210> 94

<211> 38

<212> DNA

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<400> 94

catcggcgta cgactgagtt agcatccacg tgcttgag 38

<210> 95

<211> 38

<212> DNA

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<400> 95

catcggcgta cgactgatga atcatccacg tgcttgag 38

<210> 96

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<400> 96

catcggcgta cgactgccaa gacatccacg tgcttgag 38

<210> 97

<211> 55

<212> DNA

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<400> 97

cagacgtgtg ctcttccgat ctnnnnnnnn nnaacgtgat gtggccgatg tttcg 55

<210> 98

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 98

cagacgtgtg ctcttccgat ctnnnnnnnn nnaaacatcg gtggccgatg tttcg 55

<210> 99

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 99

cagacgtgtg ctcttccgat ctnnnnnnnn nnatgcctaa gtggccgatg tttcg 55

<210> 100

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 100

cagacgtgtg ctcttccgat ctnnnnnnnn nnagtggtca gtggccgatg tttcg 55

<210> 101

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 101

cagacgtgtg ctcttccgat ctnnnnnnnn nnaccactgt gtggccgatg tttcg 55

<210> 102

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 102

cagacgtgtg ctcttccgat ctnnnnnnnn nnacattggc gtggccgatg tttcg 55

<210> 103

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 103

cagacgtgtg ctcttccgat ctnnnnnnnn nncagatctg gtggccgatg tttcg 55

<210> 104

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 104

cagacgtgtg ctcttccgat ctnnnnnnnn nncatcaagt gtggccgatg tttcg 55

<210> 105

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 105

cagacgtgtg ctcttccgat ctnnnnnnnn nncgctgatc gtggccgatg tttcg 55

<210> 106

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 106

cagacgtgtg ctcttccgat ctnnnnnnnn nnacaagcta gtggccgatg tttcg 55

<210> 107

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 107

cagacgtgtg ctcttccgat ctnnnnnnnn nnctgtagcc gtggccgatg tttcg 55

<210> 108

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 108

cagacgtgtg ctcttccgat ctnnnnnnnn nnagtacaag gtggccgatg tttcg 55

<210> 109

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 109

cagacgtgtg ctcttccgat ctnnnnnnnn nnaacaacca gtggccgatg tttcg 55

<210> 110

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 110

cagacgtgtg ctcttccgat ctnnnnnnnn nnaaccgaga gtggccgatg tttcg 55

<210> 111

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 111

cagacgtgtg ctcttccgat ctnnnnnnnn nnaacgctta gtggccgatg tttcg 55

<210> 112

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 112

cagacgtgtg ctcttccgat ctnnnnnnnn nnaagacgga gtggccgatg tttcg 55

<210> 113

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 113

cagacgtgtg ctcttccgat ctnnnnnnnn nnaaggtaca gtggccgatg tttcg 55

<210> 114

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 114

cagacgtgtg ctcttccgat ctnnnnnnnn nnacacagaa gtggccgatg tttcg 55

<210> 115

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 115

cagacgtgtg ctcttccgat ctnnnnnnnn nnacagcaga gtggccgatg tttcg 55

<210> 116

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 116

cagacgtgtg ctcttccgat ctnnnnnnnn nnacctccaa gtggccgatg tttcg 55

<210> 117

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 117

cagacgtgtg ctcttccgat ctnnnnnnnn nnacgctcga gtggccgatg tttcg 55

<210> 118

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 118

cagacgtgtg ctcttccgat ctnnnnnnnn nnacgtatca gtggccgatg tttcg 55

<210> 119

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 119

cagacgtgtg ctcttccgat ctnnnnnnnn nnactatgca gtggccgatg tttcg 55

<210> 120

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 120

cagacgtgtg ctcttccgat ctnnnnnnnn nnagagtcaa gtggccgatg tttcg 55

<210> 121

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 121

cagacgtgtg ctcttccgat ctnnnnnnnn nnagatcgca gtggccgatg tttcg 55

<210> 122

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 122

cagacgtgtg ctcttccgat ctnnnnnnnn nnagcaggaa gtggccgatg tttcg 55

<210> 123

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 123

cagacgtgtg ctcttccgat ctnnnnnnnn nnagtcacta gtggccgatg tttcg 55

<210> 124

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 124

cagacgtgtg ctcttccgat ctnnnnnnnn nnatcctgta gtggccgatg tttcg 55

<210> 125

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 125

cagacgtgtg ctcttccgat ctnnnnnnnn nnattgagga gtggccgatg tttcg 55

<210> 126

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 126

cagacgtgtg ctcttccgat ctnnnnnnnn nncaaccaca gtggccgatg tttcg 55

<210> 127

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 127

cagacgtgtg ctcttccgat ctnnnnnnnn nngactagta gtggccgatg tttcg 55

<210> 128

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 128

cagacgtgtg ctcttccgat ctnnnnnnnn nncaatggaa gtggccgatg tttcg 55

<210> 129

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 129

cagacgtgtg ctcttccgat ctnnnnnnnn nncacttcga gtggccgatg tttcg 55

<210> 130

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 130

cagacgtgtg ctcttccgat ctnnnnnnnn nncagcgtta gtggccgatg tttcg 55

<210> 131

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 131

cagacgtgtg ctcttccgat ctnnnnnnnn nncataccaa gtggccgatg tttcg 55

<210> 132

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 132

cagacgtgtg ctcttccgat ctnnnnnnnn nnccagttca gtggccgatg tttcg 55

<210> 133

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 133

cagacgtgtg ctcttccgat ctnnnnnnnn nnccgaagta gtggccgatg tttcg 55

<210> 134

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 134

cagacgtgtg ctcttccgat ctnnnnnnnn nnccgtgaga gtggccgatg tttcg 55

<210> 135

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 135

cagacgtgtg ctcttccgat ctnnnnnnnn nncctcctga gtggccgatg tttcg 55

<210> 136

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 136

cagacgtgtg ctcttccgat ctnnnnnnnn nncgaactta gtggccgatg tttcg 55

<210> 137

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 137

cagacgtgtg ctcttccgat ctnnnnnnnn nncgactgga gtggccgatg tttcg 55

<210> 138

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 138

cagacgtgtg ctcttccgat ctnnnnnnnn nncgcataca gtggccgatg tttcg 55

<210> 139

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 139

cagacgtgtg ctcttccgat ctnnnnnnnn nnctcaatga gtggccgatg tttcg 55

<210> 140

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 140

cagacgtgtg ctcttccgat ctnnnnnnnn nnctgagcca gtggccgatg tttcg 55

<210> 141

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 141

cagacgtgtg ctcttccgat ctnnnnnnnn nnctggcata gtggccgatg tttcg 55

<210> 142

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 142

cagacgtgtg ctcttccgat ctnnnnnnnn nngaatctga gtggccgatg tttcg 55

<210> 143

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 143

cagacgtgtg ctcttccgat ctnnnnnnnn nncaagacta gtggccgatg tttcg 55

<210> 144

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 144

cagacgtgtg ctcttccgat ctnnnnnnnn nngagctgaa gtggccgatg tttcg 55

<210> 145

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 145

cagacgtgtg ctcttccgat ctnnnnnnnn nngatagaca gtggccgatg tttcg 55

<210> 146

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 146

cagacgtgtg ctcttccgat ctnnnnnnnn nngccacata gtggccgatg tttcg 55

<210> 147

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 147

cagacgtgtg ctcttccgat ctnnnnnnnn nngcgagtaa gtggccgatg tttcg 55

<210> 148

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 148

cagacgtgtg ctcttccgat ctnnnnnnnn nngctaacga gtggccgatg tttcg 55

<210> 149

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 149

cagacgtgtg ctcttccgat ctnnnnnnnn nngctcggta gtggccgatg tttcg 55

<210> 150

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 150

cagacgtgtg ctcttccgat ctnnnnnnnn nnggagaaca gtggccgatg tttcg 55

<210> 151

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 151

cagacgtgtg ctcttccgat ctnnnnnnnn nnggtgcgaa gtggccgatg tttcg 55

<210> 152

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 152

cagacgtgtg ctcttccgat ctnnnnnnnn nngtacgcaa gtggccgatg tttcg 55

<210> 153

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 153

cagacgtgtg ctcttccgat ctnnnnnnnn nngtcgtaga gtggccgatg tttcg 55

<210> 154

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 154

cagacgtgtg ctcttccgat ctnnnnnnnn nngtctgtca gtggccgatg tttcg 55

<210> 155

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 155

cagacgtgtg ctcttccgat ctnnnnnnnn nngtgttcta gtggccgatg tttcg 55

<210> 156

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 156

cagacgtgtg ctcttccgat ctnnnnnnnn nntaggatga gtggccgatg tttcg 55

<210> 157

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 157

cagacgtgtg ctcttccgat ctnnnnnnnn nntatcagca gtggccgatg tttcg 55

<210> 158

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 158

cagacgtgtg ctcttccgat ctnnnnnnnn nntccgtcta gtggccgatg tttcg 55

<210> 159

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 159

cagacgtgtg ctcttccgat ctnnnnnnnn nntcttcaca gtggccgatg tttcg 55

<210> 160

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 160

cagacgtgtg ctcttccgat ctnnnnnnnn nntgaagaga gtggccgatg tttcg 55

<210> 161

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 161

cagacgtgtg ctcttccgat ctnnnnnnnn nntggaacaa gtggccgatg tttcg 55

<210> 162

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 162

cagacgtgtg ctcttccgat ctnnnnnnnn nntggcttca gtggccgatg tttcg 55

<210> 163

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 163

cagacgtgtg ctcttccgat ctnnnnnnnn nntggtggta gtggccgatg tttcg 55

<210> 164

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 164

cagacgtgtg ctcttccgat ctnnnnnnnn nnttcacgca gtggccgatg tttcg 55

<210> 165

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 165

cagacgtgtg ctcttccgat ctnnnnnnnn nnaactcacc gtggccgatg tttcg 55

<210> 166

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 166

cagacgtgtg ctcttccgat ctnnnnnnnn nnaagagatc gtggccgatg tttcg 55

<210> 167

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 167

cagacgtgtg ctcttccgat ctnnnnnnnn nnaaggacac gtggccgatg tttcg 55

<210> 168

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 168

cagacgtgtg ctcttccgat ctnnnnnnnn nnaatccgtc gtggccgatg tttcg 55

<210> 169

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 169

cagacgtgtg ctcttccgat ctnnnnnnnn nnaatgttgc gtggccgatg tttcg 55

<210> 170

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 170

cagacgtgtg ctcttccgat ctnnnnnnnn nnacacgacc gtggccgatg tttcg 55

<210> 171

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 171

cagacgtgtg ctcttccgat ctnnnnnnnn nnacagattc gtggccgatg tttcg 55

<210> 172

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 172

cagacgtgtg ctcttccgat ctnnnnnnnn nnagatgtac gtggccgatg tttcg 55

<210> 173

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 173

cagacgtgtg ctcttccgat ctnnnnnnnn nnagcacctc gtggccgatg tttcg 55

<210> 174

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 174

cagacgtgtg ctcttccgat ctnnnnnnnn nnagccatgc gtggccgatg tttcg 55

<210> 175

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 175

cagacgtgtg ctcttccgat ctnnnnnnnn nnaggctaac gtggccgatg tttcg 55

<210> 176

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 176

cagacgtgtg ctcttccgat ctnnnnnnnn nnatagcgac gtggccgatg tttcg 55

<210> 177

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 177

cagacgtgtg ctcttccgat ctnnnnnnnn nnatcattcc gtggccgatg tttcg 55

<210> 178

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 178

cagacgtgtg ctcttccgat ctnnnnnnnn nnattggctc gtggccgatg tttcg 55

<210> 179

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 179

cagacgtgtg ctcttccgat ctnnnnnnnn nncaaggagc gtggccgatg tttcg 55

<210> 180

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 180

cagacgtgtg ctcttccgat ctnnnnnnnn nncaccttac gtggccgatg tttcg 55

<210> 181

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 181

cagacgtgtg ctcttccgat ctnnnnnnnn nnccatcctc gtggccgatg tttcg 55

<210> 182

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 182

cagacgtgtg ctcttccgat ctnnnnnnnn nnccgacaac gtggccgatg tttcg 55

<210> 183

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 183

cagacgtgtg ctcttccgat ctnnnnnnnn nncctaatcc gtggccgatg tttcg 55

<210> 184

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 184

cagacgtgtg ctcttccgat ctnnnnnnnn nncctctatc gtggccgatg tttcg 55

<210> 185

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 185

cagacgtgtg ctcttccgat ctnnnnnnnn nncgacacac gtggccgatg tttcg 55

<210> 186

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 186

cagacgtgtg ctcttccgat ctnnnnnnnn nncggattgc gtggccgatg tttcg 55

<210> 187

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 187

cagacgtgtg ctcttccgat ctnnnnnnnn nnctaaggtc gtggccgatg tttcg 55

<210> 188

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 188

cagacgtgtg ctcttccgat ctnnnnnnnn nngaacaggc gtggccgatg tttcg 55

<210> 189

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 189

cagacgtgtg ctcttccgat ctnnnnnnnn nngacagtgc gtggccgatg tttcg 55

<210> 190

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 190

cagacgtgtg ctcttccgat ctnnnnnnnn nngagttagc gtggccgatg tttcg 55

<210> 191

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 191

cagacgtgtg ctcttccgat ctnnnnnnnn nngatgaatc gtggccgatg tttcg 55

<210> 192

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 192

cagacgtgtg ctcttccgat ctnnnnnnnn nngccaagac gtggccgatg tttcg 55

<210> 193

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 193

gatctg 6

<210> 194

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 194

tcaagt 6

<210> 195

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 195

ctgatc 6

<210> 196

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 196

aagcta 6

<210> 197

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 197

gtagcc 6

<210> 198

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 198

tacaag 6

<210> 199

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 199

ttgact 6

<210> 200

<211> 6

<212> DNA

<213> Artificial Sequence

<400> 200

ggaact 6

<210> 201

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 201

gatcctcaag cacgtggat 19

<210> 202

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 202

agtcgtacgc cgatgcgaaa catcggccac 30

<210> 203

<211> 5

<212> DNA

<213> Artificial Sequence

<400> 203

gatca 5

<210> 204

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 204

aatgatacgg cgaccaccga gatctacact agatcgctcg tcggcagcgt c 51

<210> 205

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 205

caagcagaag acggcatacg agatgatctg gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 206

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 206

caagcagaag acggcatacg agattcaagt gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 207

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 207

caagcagaag acggcatacg agatctgatc gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 208

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 208

caagcagaag acggcatacg agataagcta gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 209

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 209

caagcagaag acggcatacg agatgtagcc gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 210

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 210

caagcagaag acggcatacg agattacaag gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 211

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 211

caagcagaag acggcatacg agatttgact gtgactggag ttcagacgtg tgctcttccg 60

atct 64

<210> 212

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 212

caagcagaag acggcatacg agatggaact gtgactggag ttcagacgtg tgctcttccg 60

atct 64

Claims (4)

1. A method of constructing a DNA library for single cell genomic sequencing, comprising:

step S110: fragmenting DNA in the cell nucleus to obtain a fragmented cell nucleus of the DNA;

step S120: carrying out multiple rounds of marking on the fragmented DNA in the cell nuclei by adopting different sequence tags, so that the fragmented DNA in each cell nucleus is connected with a tag code consisting of the sequence tags, and the tag codes connected with the fragmented DNA in each cell nucleus are different; a kind of electronic device with high-pressure air-conditioning system

Step S130: amplifying the fragmented DNA connected with the tag codes to obtain a DNA library for sequencing a single cell genome;

the cell nucleus is derived from human cells 293T and murine cells CT26;

the step S120 includes:

grouping the fragmented cell nucleuses of the plurality of DNA, and marking the fragmented DNA in each group of the cell nucleuses by adopting different first sequence tags, so that the fragmented DNA in each group of the cell nucleuses is connected with the first sequence tags, and the first sequence tags connected with the fragmented DNA in each group of the cell nucleuses are different, thereby obtaining a plurality of groups of primary marked cell nucleuses;

mixing and grouping a plurality of groups of primary marked cell nuclei, and marking the segmented DNA in the grouped primary marked cell nuclei by adopting different second sequence tags, so that the segmented DNA in each group of primary marked cell nuclei is connected with the second sequence tags, and the second sequence tags connected with the segmented DNA in each group of primary marked cell nuclei are different to obtain a plurality of groups of secondary marked cell nuclei;

mixing and grouping a plurality of groups of secondary marked cell nuclei, and marking fragmented DNA in the grouped secondary marked cell nuclei by adopting different third sequence tags, so that the fragmented DNA of each group of secondary marked cell nuclei is connected with the third sequence tags, the third sequence tags connected with the DNA of each group of secondary marked cell nuclei are different, and a plurality of groups of tertiary marked cell nuclei are obtained, wherein the product of the number of the first sequence tags, the number of the second sequence tags and the number of the third sequence tags is larger than the number of the cell nuclei fragmented by the DNA;

the tag code of the fragmented DNA of each cell nucleus is formed by sequentially connecting the first sequence tag, the second sequence tag and the third sequence tag corresponding to the fragmented DNA of each cell nucleus;

the first sequence tag comprises a first sequence for identification, wherein the base sequence of the first sequence is selected from one of the base sequences shown in SEQ ID No. 1-SEQ ID No. 96;

the second sequence tag comprises a second sequence for identification, and the base sequence of the second sequence is selected from one of the base sequences shown in SEQ ID No. 97-SEQ ID No. 192;

the third sequence tag comprises a third sequence for identification, wherein the base sequence of the third sequence is selected from one of the base sequences shown in SEQ ID No. 193-SEQ ID No. 200;

the first sequence tag also comprises a first connecting sequence for connecting with the second sequence tag, and the base sequence of the first connecting sequence is shown as SEQ ID No. 201;

the second sequence tag also comprises a second connecting sequence used for connecting with the first sequence tag, and the base sequence of the second connecting sequence is shown as SEQ ID No. 202;

after grouping the cell nuclei in which the plurality of DNA is fragmented, marking the fragmented DNA in each group of the cell nuclei by adopting different first sequence tags, so that the fragmented DNA in each group of the cell nuclei is connected with the first sequence tags, the first sequence tags connected with the fragmented DNA of each group of the cell nuclei are different, and after the step of obtaining a plurality of groups of primary marked cell nuclei, the method further comprises the step of respectively mixing each group of primary marked cell nuclei with blocking sequences;

the base sequence of the blocking sequence is shown as SEQ ID No. 203.

2. The method of constructing a DNA library for sequencing a single cell genome according to claim 1, wherein after grouping the plurality of DNAs into the fragmented nuclei, the fragmented DNAs in each of the plurality of the nuclei are labeled with different first sequence tags such that the first sequence tags are attached to the fragmented DNAs in each of the plurality of the nuclei, and the first sequence tags attached to the fragmented DNAs in each of the plurality of the nuclei are different, the step of obtaining a plurality of sets of primary labeled nuclei comprises:

grouping the segmented cell nuclei of the plurality of DNA, mixing the segmented cell nuclei with different first sequence tags, and incubating the mixture to obtain a plurality of groups of pre-ligation liquid containing the different first sequence tags; a kind of electronic device with high-pressure air-conditioning system

And adding DNA ligase into each group of the pre-ligation liquid, and then incubating to obtain a plurality of groups of primary marked cell nuclei.

3. The method for constructing a DNA library for single cell genomic sequencing according to claim 1 or 2, wherein the 5 'end of the first sequence is linked to a phosphate group and the 5' end of the second sequence is linked to biotin.

4. The method of constructing a DNA library for single cell genomic sequencing according to claim 1, wherein the step S120 comprises:

grouping a plurality of cell nuclei with fragmented DNA, and marking the fragmented DNA in each group of the cell nuclei by adopting different first sequence tags, so that the fragmented DNA in each group of the cell nuclei is connected with the first sequence tags, the first sequence tags connected with the fragmented DNA of each group of the cell nuclei are different, and a plurality of groups of primary marked cell nuclei are obtained, wherein the first sequence tags are sequence tags;

mixing and grouping a plurality of groups of primary marked cell nuclei, and marking the segmented DNA in the grouped primary marked cell nuclei by adopting different second sequence tags, so that the segmented DNA in each group of primary marked cell nuclei is connected with the second sequence tags, the second sequence tags connected with the segmented DNA of each group of primary marked cell nuclei are different, and a plurality of groups of secondary marked cell nuclei are obtained, wherein the second sequence tags are sequence tags;

uniformly grouping a plurality of groups of secondary marked cell nuclei after mixing to obtain a plurality of groups of to-be-lysed liquid, wherein the fragmented DNA in each cell nucleus in the to-be-lysed liquid is connected with a tag code consisting of the first sequence tag and the second sequence tag, and the tag codes connected with the fragmented DNA of each cell nucleus are different; or the probability of the same label code of the fragmented DNA connection of the cell nuclei in the same group of the to-be-lysed liquid is less than 5%;

and (3) splitting one group of the to-be-split solution to release fragmented DNA connected with a first sequence tag and a second sequence tag in each cell nucleus in the to-be-split solution, then fragmenting the fragmented DNA connected with the first sequence tag and the second sequence tag by a fragment technology, and connecting a database building joint containing a third sequence tag to obtain a plurality of fragmented DNA connected with tag codes consisting of the first sequence tag, the second sequence tag and the third sequence tag, wherein the tag codes of the fragmented DNA connected with the third sequence tag are different.

Images