HK1168627B - Dna (deoxyribonucleic acid) index library building method based on pcr (polymerase chain reaction) - Google Patents

Abstract

The present invention designs a unique 161 tag sequence with a length of 8bp, and embeds the tags into DNA PCR primers to form DNA PCR tag primers, which can be introduced into the tag sequence through PCR reaction. The present invention has successfully established a method for building a DNA tag library and applied it to solexa DNA sequencing.

Description

DNA label library construction method based on PCR

Technical Field

The invention relates to the technical field of DNA library construction, in particular to the technical field of DNA label library construction, and particularly relates to a DNA label library construction method based on PCR. In addition, the invention also relates to a label technology and a method for realizing library construction of a plurality of samples in the same reaction system. The method of the invention is particularly suitable for second generation sequencing techniques, in particular solexa sequencing techniques.

Background

The Solexa DNA Sequencing platform provided by Illumina company can add four kinds of nucleotides with fluorescent labels in one reaction, adopts Sequencing by synthesis and Sequencing (SBS), and has the characteristics of less required sample amount, high flux, high accuracy, simple and easy-to-operate automation platform, powerful function and the like [1-4 ]. Library construction first requires end repair of the target fragment, ligation of an "A" base at the 3 'end of the target fragment, ligation of the target fragment with an "A" base at the 3' end to a DNA adaptor (also known as adapter), amplification of the target fragment by PCR reaction, and recovery of the library of target fragments containing the DNA adaptor, as shown in FIG. 1. The target fragment library is hybridized with a DNA joint on a sequencing chip, amplified by bridge PCR, and finally synthesized and sequenced. During each cycle, fluorescently labeled nucleosides and polymerase are added to the single molecule array. The first base pairing of the complementary nucleoside and nucleotide fragments is added to the primer by the enzyme. Excess nucleosides were removed. Thus, each single-stranded DNA molecule is extended by the pairing of complementary bases, and laser light of a specific wavelength for each base excites the label of the bound nucleoside, which releases fluorescence, and the resulting fluorescent signal is translated into a base sequence. At present, the DNA library construction method can be applied to various research fields such as De Novo sequencing of genome, genome re-sequencing, transcriptome sequencing, epigenome sequencing and the like according to requirements.

Based on the above library construction method, the library construction method of DNA tags (also called index) was also introduced by illumina, Inc., as shown in FIG. 2. In the DNA tag library construction process, 3 PCR tag primers are used in the PCR process, and a DNA tag library is constructed by introducing tags through PCR [5 ]. In patent applications WO2005068656A1 and WO2008093098A2, a method is disclosed in which the source of a nucleic acid sample is tagged with a tag sequence that allows for mixed sequencing of the sample, and a specific nucleotide sequence (i.e., tag sequence) can be introduced into the library by PCR through the process of PCR, with the PCR tag primer sequences being shown in Table 1. These tagged libraries can be mixed arbitrarily as required, then sequenced by solexa sequencer, and finally the data sorted by tag-tagged sequence.

However, the method for preparing the tag library provided by the illumina company has some defects: firstly, the illumina company only provides 12 tag sequences with the length of 6bp, the number of tags is small, and the mixed sequencing of a large number of samples cannot be realized along with the increase of the sequencing flux of solexa, which is a huge defect; second, the tag library construction method provided by illumina company introduces tag sequences into a target fragment library through PCR reaction, 3 PCR primers are required to amplify the target fragment (two common primers and one PCR tag primer, as shown in table 1), and the PCR amplification efficiency is not high.

Third, in the tag library construction method provided by illumina, the linker does not contain a tag sequence, each tag library needs to be introduced with a tag sequence through a PCR reaction, then each tag library needs to be cut and recovered, and then the target fragment libraries of the tags after being cut and recovered are mixed, which not only wastes time and labor, but also has high cost.

Therefore, the sequence of the label and the label introducing method are optimized and improved, so that the label introducing efficiency is improved, the number of the label sequences is increased, the requirement of high-flux library construction can be met, the current situation that the sequencing flux is continuously improved is adapted, the capacity of a sequencing instrument is fully utilized, and the sequencing cost is reduced.

TABLE 1 tag sequences and corresponding PCR tag primer sequences provided by illumina

Disclosure of Invention

Based on the DNA tag library preparation method provided by the currently available solexa sequencing platform of the illumina company, the invention improves tag sequences, designs unique tag sequences with the length of 8bp respectively, can introduce the tag sequences through PCR reaction respectively, successfully establishes a library construction method of the DNA tag library, is applied to solexa DNA sequencing, improves the preparation efficiency of the DNA tag library, increases the sequencing flux of DNA samples, and reduces the solexa sequencing cost of single samples.

The design of the tag firstly needs to consider the problems of identifiability and identification rate between tag sequences, then needs to consider the balance problem of GT and AC base content of each site after the tag sequences are mixed, and finally considers the repeatability and accuracy of data output. In the process of designing the label, the invention fully considers the factors and simultaneously avoids the occurrence of more than 3 or 3 continuous bases in the nucleic acid sequence of the label, thus reducing the error rate of the sequence in the synthesis process or the sequencing process. Meanwhile, the reduction of PCR amplification efficiency caused by the hairpin structure formed by the tag primer is avoided as much as possible.

The invention optimizes the DNA joint sequence provided by the illumina, designs the label into a special sequence with the length of 8bp, and optimizes 3DNA label PCR primers provided by the illumina company into two PCR primers to be introduced into the library building method of the label. Meanwhile, the library building method for introducing the label through 3DNA label PCR primers provided by the illumina company is optimized to introduce the label through two PCR primers, and compared with the DNA label primers of the illumina company, the optimized label primers improve the efficiency of PCR amplification reaction and the identification efficiency of the label sequence. FIG. 3 shows a DNA tag library construction flowchart of the illiminia company, and FIG. 4 shows an optimized DNA tag library construction experiment flowchart.

The invention designs a specific tag nucleic acid sequence with the length of 8bp based on a Solexa Paired End sequencing platform provided by the currently available illumina company. By testing the amplification efficiency of the DNA label PCR primers containing the specific label nucleic acid sequence and the recognition rate of the label nucleic acid sequence, 161 DNA label sequences (shown in Table 2, DNA label sequences of 8 bp) with the length of 8bp and DNA PCR label primers are optimized and screened. The difference between these tags of 8bp in length is 4 bases, i.e. at least 4 bases in sequence. When any one of 8 bases of the label has sequencing error or synthesis error, the final identification of the label is not influenced.

TABLE 2 DNA tag (indexN) sequence of 8bp in length

index number	Sequence of	index number	Sequence of	index number	Sequence of
						Index1	CATTGCTT	Index55	TACAGGCC	Index109	TCCGACGG
Index2	TTCGGATT	Index56	GTTAAGCC	Index110	GCAGGCAT
						Index3	TCATCATT	Index57	TAATTACC	Index111	GCCAGCGA
Index4	GCTCCTGT	Index58	ATAACACC	Index112	CACACTGG
						Index5	AGCTCGGT	Index59	CGTAGGAC	Index113	GGCCTCGC
Index6	CAACAGGT	Index60	CTCTCGAC	Index114	GGCGCGCA
						Index7	TTCAAGGT	Index61	CTACGCAC	Index115	CGCCACCT
Index8	CCTAACGT	Index62	AGGTTAAC	Index116	CATGCGGC
						Index9	CACGTAGT	Index63	GTTGCAAC	Index117	GGCAACAG
Index10	GTAAGAGT	Index64	CTCAATTA	Index118	CGGTATCA
						Index11	TACCTTCT	Index65	CAAGTCTA	Index119	CGGCCAAT
Index12	AAGTCTCT	Index66	ACAACCTA	Index120	AGCCGTCC
						Index13	AGAGATCT	Index67	CTACCATA	Index121	ACAGAGTG
Index14	CCAGCGCT	Index68	GACACATA	Index122	ACGCAGCC
						Index15	ATGAACCT	Index69	AGATAATA	Index123	GAGCTGAC
Index16	ACCAGACT	Index70	CGCGGTGA	Index124	TGATGGCT
						Index17	CTATAACT	Index71	TACTATGA	Index125	TGAATCAT
Index18	GCGGAACT	Index72	TTGTTGGA	Index126	TGACAGAC

Index19	CTAGTTAT	Index73	AGTGAGGA	Index127	GTGGTCGT
						Index20	TCTTATAT	Index74	ATCGCCGA	Index128	GCGTGGAG
Index21	GAATCGAT	Index75	CTTATAGA	Index129	ACTTCCGC
						Index22	AATAAGAT	Index76	CCATGAGA	Index130	ACATGTAC
Index23	TATGCCAT	Index77	TCACCTCA	Index131	CCGGCTAA
						Index24	ATTCTAAT	Index78	ACCTTGCA	Index132	CGATCCTG
Index25	TAATGTTG	Index79	ATACTCCA	Index133	GACGATAT
						Index26	GTTACTTG	Index80	GTTCGACA	Index134	CCTGGCCA
Index27	ATTCACTG	Index81	CATCATAA	Index135	AAGACGTC
						Index28	ATCATATG	Index82	CACATGAA	Index136	GCTCTCTA
Index29	GCTTAATG	Index83	ATGAGGAA	Index137	AGCGTGTC
						Index30	GGATATGG	Index84	TCCTCCAA	Index138	CCGTTGTT
Index31	CTTGATGG	Index85	TTAGACAA	Index139	TTGCTACG
						Index32	AAGATCGG	Index86	GTCCAGAA	Index140	TGTAACCA
Index33	TTAACCGG	Index87	ATCTATCG	Index141	TGTGTTAA
						Index34	CTAAGTCG	Index88	TTACTGTT	Index142	GATAGCCG
Index35	TATTCGCG	Index89	ACACGCGG	Index143	TAACACCG
						Index36	GAAGCACG	Index90	TATCCAGA	Index144	AGTAGTTA
Index37	TCCAGTAG	Index91	TAGGAATA	Index145	GTCTGCCT
						Index38	TTGTCTAG	Index92	GAACGTGA	Index146	GGAGTAGA
Index39	AGCGCTAG	Index93	CCGCACAG	Index147	TGCGCAGC
						Index40	CCTGTGAG	Index94	ATTGCGTT	Index148	TGCCTATA
Index41	CAACTAAG	Index95	TCGTAAGC	Index149	TGCTAGTG
						Index42	ATAGGAAG	Index96	CCGTCACG	Index150	CCGAGCTC
Index43	ACTACAAG	Index97	GCGAAGTA	Index151	CGGATTAG
						Index44	GATGGTTC	Index98	GGACTGCG	Index152	CGGACGGA
Index45	CCACATTC	Index99	GAGCATTG	Index153	GACTGAGG
						Index46	TCTTGGTC	Index100	TCGCCGTG	Index154	GTGTGTTA
Index47	CGAGGATC	Index101	CAGCGGCG	Index155	CTCGTCCG
						Index48	AGTCCATC	Index102	AAGGATGC	Index156	TGGAGAGG
Index49	CACTAATC	Index103	GCAATGGC	Index157	TGGAATTC
						Index50	TAAGGCGC	Index104	GTATTCTC	Index158	TTGGCGCC
Index51	AATAGAGC	Index105	GTCATTAC	Index159	GCCTTAAT
						Index52	ACTGTTCC	Index106	ATCCAAGC	Index160	AAGCGATT
Index53	CTTCCTCC	Index107	GGTATACT	Index161	AACCGCAA
						Index54	GCGACTCC	Index108	TTGCGTGC

Schematic representation of hairpin structure formed by the predicted DNA PCR tag primer and secondary linker structure of itself using PrimerSelect software from Lasergene. Wherein [ ST _ Hairpin ] Score represents a card issuance Score; [ AD _ Self _ Extend _ Dimer ] Score represents Self-extended Dimer Score; [ ST _ Self _ Dimer ] Score represents the Self-Dimer Score.

Table 3: predicting the hairpin structure formed by the DNA PCR label primer and the secondary structure of the DNA PCR label primer, and scoring of different DNA PCR label primers.

Drawings

FIG. 1: schematic diagram of conventional DNA library construction process provided by Ilumina.

FIG. 2: schematic diagram of conventional DNA tag library creation process provided by illumina corporation.

FIG. 3: DNA tag library creation flow chart of the company illumina.

FIG. 4: and (5) establishing a library experimental flow chart of the optimized DNA label.

FIG. 5: the ratio of 1mismatch/0mismatch (1mismatch/0mismatch) of DNA tag primers with a length of 8bp is about 2% for most 1mismatch/0mismatch, and about 4% for a few, and better data results can be obtained because the ratio of 1mismatch/0mismatch is less than 5%, so 161 DNA tag primers with a length of 8bp pass the test.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

In one aspect, the invention provides a set of DNA tags comprising or consisting of: at least 10, or at least 20, or at least 30, or at least 40, at least 50, or at least 60, or at least 70, or at least 80, or 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or all 161 of the 161 DNA tags shown in Table 2, or DNA tags differing therefrom by 1 base,

the DNA tags preferably comprise at least the 161 DNA tags shown in Table 2, DNAdex 1-DNA Index10, or DNA Index 11-DNA Index20, or DNAdex 21-DNA Index30, or DNA Index 31-DNA Index40, or DNAdex 41-DNA Index50, or DNA Index 51-DNA Index60, or DNAdex 61-DNA Index70, or DNA Index 71-DNA Index80, or DNAdex 81-DNA Index90, or DNA Index 91-DNA Index100, or DNAIndex 101-DNA Index110, or DNA Index 111-DNA Index120, or DNAIndex 121-DNA Index130, or DNA Index 131-DNA Index140, or DNAIndex 141-DNA Index150, or DNA Index 151-DNA Index161, or any combination of two or more thereof.

In one embodiment of the invention, a1 base difference as described in relation to said DNA tag comprises a substitution, addition or deletion of 1 base in the tag.

In a specific embodiment of the invention, the invention provides the use of said DNA tags for the construction of a DNA tag library, wherein the DNA tag adaptors of the DNA tag library comprise said DNA tags at the 3 'end, thereby constituting the respective corresponding DNA tag adaptors, which are preferably used as the 3' adaptors of the DNA tag library.

In a specific embodiment of the invention, in the uses provided by the invention, the DNA tag is inserted into the 3 ' end of the DNA tag adaptor or is linked to the 3 ' end of the DNA adaptor, preferably into the 3 ' end of the DNA tag adaptor, with or without a linker; more preferably 1 base from the 3' end in the DNA tag adaptor. The linker is a 1-6 nucleotide sequence, preferably a 1-3 nucleotide sequence.

In one embodiment of the invention, the invention provides a DNA tag library constructed using said DNA tags.

In another aspect the present invention provides a set of DNA PCR tag primers comprising a DNA tag as described above, wherein the DNA PCR tag primers comprise at the 3' end a tag as described above, said set of said DNA PCR tag primers comprising or consisting of: at least 10, or at least 20, or at least 30, or at least 40, at least 50, or at least 60, or at least 70, or at least 80, or 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or all 161 of the 161 DNA PCR tag primers shown in Table 4, or DNA PCR tag primers differing by 1 base from the DNA tag sequence they comprise,

the DNA PCR-tagged primers preferably at least comprise DNA PCR index1 primer-DNA PCR index10primer, or DNA PCR index11 primer-DNA PCR index20primer, or DNA PCR index21 primer-DNA PCR index30primer, or DNA PCR index31 primer-DNA PCR index40primer, or DNA PCR index41 primer-DNA PCR index50primer, or DNA PCR index51 primer-DNA PCR index60primer, or DNA PCR index61 primer-DNA PCR index70primer, or DNA PCR index 71-DNA PCR index80primer, or DNA PCR index81 primer-DNA PCR index90, or DNA PCR index 91-DNA primer-DNA index100primer, or DNA PCR index 101-DNA primer, DNA PCR index 110-DNA PCR index, DNA PCR index 141-DNA PCR index or DNA PCR index111primer, DNA PCR index 141-DNA primer, or DNA PCR index111 primer-DNA primer, or DNA PCR index 111-DNA primer, PCR index 141-DNA primer, DNA primer or DNA PCR index111 primer-DNA primer, or DNA PCR index151primer to DNA PCR index161primer, or a combination of any two or more thereof.

In one embodiment of the invention said 1 base difference in said DNA PCR tag primer comprises a substitution, addition or deletion of 1 base in the tag.

In a specific embodiment of the present invention, the present invention provides the use of the DNAPCR tag primers described above for the construction of a DNA tag library, preferably the DNA PCR tag primers are used as downstream primers of a DNA tag library.

In one embodiment of the invention, the invention provides a DNA tag library constructed by the DNA PCR tag primers described above.

In another aspect, the present invention provides a method for constructing a tag library, wherein the method is characterized in that a DNA PCR tag primer comprising a tag is used to construct the tag library.

In one embodiment of the present invention, there is provided a method for constructing a tag library, the method comprising:

1) providing n DNA samples, n being an integer and 1. ltoreq. n.ltoreq.161, preferably n being an integer and 2. ltoreq. n.ltoreq.161, from all eukaryotic and prokaryotic DNA samples, including but not limited to human DNA samples;

2) breaking the genome DNA, wherein the breaking method includes but is not limited to an ultrasonic breaking method, and preferably the broken DNA bands are concentrated to about 200 bp;

3) repairing the tail end;

4) adding an "A" base at the 3' end of the DNA fragment;

5) connecting a DNA adaptor;

6) performing gel recovery and purification on the ligation product obtained in the step 5), preferably performing electrophoresis through 2% agarose gel and recovering, and mixing the recovery products of the DNA samples together;

7) and (3) performing PCR reaction by using the mixture of the recovered products in the step 6) as a template under the condition suitable for amplifying the target nucleic acid, and performing gel recovery and purification on the PCR product, preferably recovering a 280-300 bp target fragment.

In one embodiment of the present invention, the primers used in the PCR reaction of step 7) in the method are as follows:

the upstream Primers were PE PCR Primers 1.0:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT；

the downstream primer is a DNA PCR tag primer comprising or consisting of: at least 10, or at least 20, or at least 30, or at least 40, at least 50, or at least 60, or at least 70, or at least 80, or 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or all 161 of the 161 DNA PCR tag primers shown in Table 4, or DNA PCR tag primers differing by 1 base from the DNA tag sequence they comprise,

the DNA PCR-tagged primers preferably at least comprise DNA PCR index1 primer-DNA PCR index10primer, or DNA PCR index11 primer-DNA PCR index20primer, or DNA PCR index21 primer-DNA PCR index30primer, or DNA PCR index31 primer-DNA PCR index40primer, or DNA PCR index41 primer-DNA PCR index50primer, or DNA PCR index51 primer-DNA PCR index60primer, or DNA PCR index61 primer-DNA PCR index70primer, or DNA PCR index 71-DNA PCR index80primer, or DNA PCR index81 primer-DNA PCR index90, or DNA PCR index 91-DNA primer-DNA index100primer, or DNA PCR index 101-DNA primer, DNA PCR index 110-DNA PCR index, DNA PCR index 141-DNA PCR index or DNA PCR index111primer, DNA PCR index 141-DNA primer, or DNA PCR index111 primer-DNA primer, or DNA PCR index 111-DNA primer, PCR index 141-DNA primer, DNA primer or DNA PCR index111 primer-DNA primer, or DNA PCR index151primer to DNA PCR index161primer, or a combination of any two or more thereof.

In one embodiment of the invention, the 1 base difference in the method provided by the invention comprises substitution, addition or deletion of 1 base in the tag.

In one embodiment of the present invention, the DNA adaptor used in step 5) of the method is PE index Adapters:

5’Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

5’TACACTCTTTCCCTACACGACGCTCTTCCGATCT。

in a further aspect of the invention there is provided a library of tags constructed by the method described above.

Paired End DNA oligonucleotide sequence:

PE index Adapters

5’Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

5’TACACTCTTTCCCTACACGACGCTCTTCCGATCT

PE PCR Primers 1.0

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

TABLE 4DNA PCR tag primers (DNA PCR indexN primer)

Main experimental apparatus and reagent

Example 1: DNA label experiment library building method

1.1DNA fragmentation

Human whole blood genomic DNA (5 ug) was disrupted for 6 minutes using a Covaris disrupter (parameter settings: Duty cycle-20%; Intensity-5.0; Bursts persecuted-200; Duration-40 seconds; Mode-Frequency scanning; Power-33-34W; Temperature-5.5 to 6 ℃ C.), so that the major bands displayed in agarose electrophoresis were concentrated at around 200bp [5 ].

1.2 end repair

The reaction mixture was prepared according to the following ratio:

DNA template 35. mu.L

50 μ L of T4 DNA ligase buffer

dNTPs mixture 4. mu.L

T4 DNA polymerase 5. mu.L

Klenow DNA polymerase 1. mu.L

T4 Polynucleotide kinase 5. mu.L

Total volume 100. mu.L

The comfortable constant temperature mixer was adjusted to 20 ℃ for 30min, then purified with a QIAquick PCR purification kit, and finally the samples were dissolved in 32. mu.L EB solution.

1.3DNA fragment 3' end plus "A" base

The reaction mixture was prepared according to the following ratio:

32. mu.L of DNA after end repair

Klenow enzyme buffer 5. mu.L

dATP(1mM) 10μL

Klenow enzyme (3 'to 5' exonuclease Activity) 3. mu.L

Total volume 50. mu.L

The temperature of a comfortable constant-temperature mixer is adjusted to 37 ℃, the reaction is carried out for 30min, then a MiniElute PCR purification kit is used for purification, and finally, the sample is dissolved in 10 mu L of EB solution.

1.4 ligation of DNA linkers

The reaction mixture was prepared according to the following ratio:

DNA 10μL

25. mu.L of T4 DNA ligase buffer

PE index Adapters 10μL

T4 DNA ligase 5. mu.L

Total volume 50. mu.L

Adjusting a comfortable constant-temperature mixer to 20 ℃, reacting for 15min, purifying by using a QIAquick PCR purification kit, and finally dissolving the sample in 30 mu L of EB solution

1.5 gel recovery purification of ligation products

Carrying out electrophoretic separation on the ligation products in 2% agarose gel; the band cut of the fragment of interest was then transferred to an Eppendorf tube. Gel purification was recovered using QIAquick gel purification kit and the recovered product was dissolved in 20. mu.L EB solution.

1.6PCR introduction of tag linker

And (3) PCR reaction: the reaction mixture was prepared according to the following reaction system, and the reagents were placed on ice.

Gel recovery of purified DNA 10. mu.L

Phusion DNA polymerase 25. mu.L

PE PCR Primers 1.0 1μL

DNA PCR tag primer 1. mu.L

ddH₂O 13μL

Total volume 50. mu.L

Note: for each DNA sample, the DNA PCR tag primer used may be any one of the DNA PCR tag primers (DNA PCR indexN primers) shown in Table 4 (Table 4),

PCR reaction conditions

98℃ 30s

72℃ 5min

Storing at 4 deg.C

1.7 gel recovery purification of PCR products

The PCR product was electrophoretically separated in 2% agarose gel, the target fragment was recovered by cleavage, gel-purified and recovered using QIAquick gel purification kit, and the recovered product was dissolved in 30. mu.L of ElutionBuffer.

1.8 detection of DNA preparation

1) Library yields were measured using an Agilent 2100 Bioanalyzer.

2) Library yield was quantified using QPCR.

As shown in the statistics of the Solexa sequencing results in FIG. 5, the proportion of 1 mismatching (mismatch)/0 mismatching is controlled below 5%, and most of the mismatching is controlled below 3%. 24362092 sequences are totally detected, wherein 23099149 sequences exist in the sequences (0mismatch) of which the tags are completely matched, 460238 sequences exist in the sequences of which 1 wrong base appears in tag sequencing, namely the proportion of the recognized tags is 96.7%, and the requirement of building a base of the solexa DNA tags can be met.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Reference to the literature

1、Paired-End sequencing User Guide；illumina part#1003880

2、Preparing samples for ChIP sequencing for DNA；illuminapart#11257047 Rev.A；

3、mRNA sequencing sample preparation Guide；illuminapart#1004898 Rev.D

4、Preparing 2-5kb samples for mate pair library sequencing；illumina part#1005363 Rev.B；

5、Preparing samples for multiplexed Paired-End sequencing；illumina part#1005361 Rev.B。

Claims (27)

1. A group of DNA PCR label primers,

the DNA PCR label primers at least comprise DNA PCR index1primer to DNA PCR index10primer in 161 DNA PCR label primers shown in Table 4.

2. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex11 primer-DNA PCR index20 primer.

3. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex21 primer-DNA PCR index30 primer.

4. The DNA PCR-tagged primers of claim 1, further comprising a DNA PCRindex31 primer-DNA PCR index40 primer.

5. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex41 primer-DNA PCR index50 primer.

6. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex51 primer-DNA PCR index60 primer.

7. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex61 primer-DNA PCR index70 primer.

8. The DNA PCR-tagged primers of claim 1, further comprising the DNA PCRindex71 primer-DNA PCR index80 primer.

9. The DNA PCR-tagged primers of claim 1, further comprising the DNA PCRindex81 primer-DNA PCR index90 primer.

10. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex91 primer-DNA PCR index100 primer.

11. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex101 primer-DNA PCR index110 primer.

12. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex111 primer-DNA PCR index120 primer.

13. The DNA PCR-tagged primers of claim 1, further comprising the DNA PCRindex121 primer-DNA PCR index130 primer.

14. The DNA PCR tag primer of claim 1, further comprising DNA PCRindex131 primer-DNA PCR index140 primer.

15. The DNA PCR-tagged primers of claim 1, further comprising DNA PCRindex141 primer-DNA PCR index150 primer.

16. The DNA PCR tag primer of claim 1, further comprising DNA PCRindex151primer to DNA PCR index161 primer.

17. Use of the DNA PCR tag primers of any one of claims 1-16 for constructing a DNA tag library.

18. The use of claim 17, wherein the DNA PCR tag primer is used as a downstream primer for a DNA tag library.

19. A DNA tag library constructed by the DNA PCR tag primers of any one of claims 1-16.

20. A method of constructing a tag library, the method characterized by using DNA PCR tag primers comprising tags to construct a tag library comprising:

1) providing n DNA samples, n being an integer and 1. ltoreq. n.ltoreq.161, from all eukaryotic and prokaryotic DNA samples;

2) breaking the genome DNA, wherein the breaking method comprises an ultrasonic breaking method;

3) repairing the tail end;

4) adding an "A" base at the 3' end of the DNA fragment;

5) connecting a DNA adaptor;

6) performing gel recovery and purification on the ligation products obtained in the step 5), and mixing the recovery products of the DNA samples together;

7) PCR reaction, using the mixture of the recovered products of the step 6) as a template, carrying out PCR amplification under the condition suitable for amplifying the target nucleic acid, and carrying out gel recovery and purification on the PCR product;

wherein the primers used in the PCR reaction of step 7) are as follows:

the upstream primers were PE PCR primers 1.0:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT；

the downstream primer is the DNA PCR tag primer of any one of claims 1-16.

21. The method of claim 20, wherein n is an integer and 2 ≦ n ≦ 161.

22. The method of claim 20, wherein the DNA sample is from a human DNA sample.

23. The method according to claim 20, wherein in step 2), the fragmented DNA bands are concentrated to about 200 bp.

24. The method of claim 20, wherein the DNA adaptor used in step 5) is a PE index Adapters:

5’Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCAC

5’TACACTCTTTCCCTACACGACGCTCTTCCGATCT。

25. the method of claim 20, wherein in step 6), the electrophoresis is performed by 2% agarose gel and recovered.

26. The method of claim 20, wherein in step 7), 280-300 bp of the target fragment is recovered.

27. A library of tags constructed by the method of any one of claims 20-26.