CN102409045B - Tag library constructing method based on DNA (deoxyribonucleic acid) adapter connection as well as used tag and tag adapter - Google Patents

Abstract

The present invention is based on the DNA tag library preparation method provided by Illumina's solexa sequencing platform, designed a unique tag sequence with a length of 6 bp, embedded the tag into the DNA adapter, and introduced the tag sequence by connecting the DNA adapter to successfully establish the DNA tag Library construction method, and applied to solexa DNA sequencing.

Description

A tag library construction method based on DNA adapter ligation and the tags and tag adapters used therefor

technical field

The invention relates to the technical field of nucleic acid sequencing, in particular to a method for preparing a DNA tag library. In addition, the present invention also relates to labeling technology and a method for constructing a label library in the same reaction system for multiple samples. The method of the present invention is particularly suitable for the second generation sequencing technology, especially the solexa sequencing technology.

Background technique

The Solexa DNA sequencing platform provided by Illumina can simultaneously add four kinds of fluorescently labeled nucleotides in one reaction, and adopts Sequencing By Synthesis (SBS), which requires less sample volume, high throughput, high Accuracy, has the characteristics of simple and easy-to-operate automation platform and powerful functions [1-4]. Library construction first needs to carry out end repair on the target fragment, connect the "A" base at the 3' end of the target fragment, connect the target fragment with the A base at the 3' end to a DNA adapter (also called an adapter), and pass PCR The reaction amplifies the target fragment, and finally recovers the target fragment library containing the DNA linker, as shown in Figure 1. The target fragment library is hybridized with the DNA adapter on the sequencing chip, amplified by bridge PCR, and finally sequenced while being synthesized. During each cycle, fluorescently labeled nucleosides and polymerase are added to single-molecule arrays. Complementary nucleosides and the first base pairing of nucleotide fragments are enzymatically added to the primer. Excess nucleosides are removed. In this way, each single-stranded DNA molecule is extended through the pairing of complementary bases, and the laser of a specific wavelength for each base excites the label of the bound nucleoside, which will release fluorescence, and finally collect the fluorescent signal for translation into a base sequence. At present, this DNA library construction method can be used in various research fields according to the needs, such as genome De Novo sequencing, genome resequencing, transcriptome sequencing and epigenome sequencing, etc.

Based on the above-mentioned library construction method, Illumina Company also introduced a DNA tag (also called index) library construction method, as shown in FIG. 2 . In the DNA label library construction process, the PCR process uses 3 PCR label primers, and the DNA label library is constructed by introducing labels through PCR [5]. Patent applications WO2005068656A1 and WO2008093098A2 disclose a method of using tag sequences to mark the source of nucleic acid samples so that samples can be mixed and sequenced, and a specific nucleotide sequence (tag sequence) can be introduced into the library by PCR , See Table 1 for the sequences of PCR index primers. These tagged libraries can be arbitrarily mixed according to requirements, and then sequenced by a solexa sequencing instrument, and finally the data are classified according to the tag sequence.

Table 1 The index (index) sequence provided by illumina company and the corresponding PCR index primer (indexN PCR primer) sequence

However, there are some defects in the method for preparing the tag library provided by Illumina: first, at present, Illumina only provides 12 tag sequences with a length of 6 bp, and the number of tags is small. Mixed sequencing of samples will be a huge defect; second, the current label library construction method provided by Illumina is to import the label sequence into the target fragment library through PCR reaction, which requires 3 PCR primers to amplify the target fragment (two common primer and a label PCR primer, as shown in Table 1), and the PCR amplification efficiency is not high. Third, the linker in the label library construction method provided by illumina does not contain the label sequence. Each label library needs to pass a PCR reaction to import the label sequence, and then for each label library, it needs to be cut and recovered, and then the glue is recovered. It is not only time-consuming and labor-intensive, but also expensive.

Therefore, only by optimizing and improving the tag sequence and tag introduction method to improve the efficiency of tag introduction and expand the number of tag sequences can we meet the requirements of high-throughput library construction and adapt to the current situation of increasing sequencing throughput. The capacity of the sequencing instrument is fully utilized to reduce the cost of sequencing.

Contents of the invention

The present invention embeds tags into DNA adapters, constructs DNA tag libraries by connecting DNA tag adapters, mixes products connected with DNA tag adapters, and completes the construction of all tag libraries that need to be mixed in one PCR reaction. It can not only improve the sequencing throughput of current DNA samples, but also improve the efficiency of library preparation and the recognition rate of tags, greatly reducing the sequencing cost of a single library.

In view of the shortcomings of the DNA tag library preparation method provided by the solexa sequencing platform of Illumina, the present invention improves the preparation method of the DNA tag library, and designs a unique tag sequence with a length of 6 bp and a linker containing the tag sequence. By including the tag The connection of the DNA adapter of the sequence to introduce the tag sequence, successfully established the library construction method of the DNA tag library, and applied it to solexa DNA sequencing, which improved the efficiency of DNA tag library preparation and increased the sequencing throughput of DNA samples. Reduced the cost of Solexa sequencing for a single sample.

In the present invention, the label design first needs to consider the identifiability and recognition rate between the label sequences, and then needs to consider the balance of the GT and AC base content of each site after the label sequence is mixed, and finally consider the data Repeatability and accuracy of output. In the process of designing tags, the present invention fully considers the above factors, and at the same time avoids 3 or more consecutive identical bases in the nucleic acid sequence of the tag, which can reduce sequence errors during synthesis or sequencing Rate. At the same time, try to avoid the formation of hairpin structure by the DNA tag adapter itself, so as to improve the connection efficiency of the DNA tag adapter.

The present invention optimizes the DNA linker sequence provided by illumina, introduces a tag sequence into the linker, and introduces the tag sequence into the target library through the connection of the DNA tag linker. In the PCR reaction after adapter ligation, there is no need to use additional label PCR primers, thereby saving the step of primer synthesis, reducing the difficulty of PCR reaction, and improving the specificity of PCR reaction. So far, there are no related reports on the library construction method and tag sequence introduced by these DNA tag adapters. Compared with Illumina's DNA adapters, the optimized DNA tag adapters have improved the efficiency of adapter ligation, and improved the recognition efficiency of tag sequences and the number of tags. Figure 3 shows the flow chart of Illumina's DNA tag library construction, and Figure 4 shows the experimental flow chart of the optimized DNA tag library construction method based on adapter ligation.

The present invention is based on the Solexa Paired End sequencing platform currently provided by Illumina, and designs a specific tag nucleic acid sequence with a length of 6 bp. By testing the amplification efficiency of DNA tag PCR primers and the recognition rate of tag nucleic acid sequences, 67 DNA tag sequences with a length of 6 bp (such as Table 2, 6 bp DNA tag sequence DNAIndex-N) and DNA tag adapters were optimized and screened Sequence (DNA Index-NF/R_adapter). The difference between these tags with a length of 6bp is at least 3 bases, that is, at least 3 bases are different in sequence. When any one of the 6 bases of the tag has a sequencing error or a synthesis error, it will not affect the final identification of the tag.

Table 2 is the forward and reverse sequences (DNA Index-NF_adapter and DNA Index-NR_adapter) of the DNA tag sequence (DNA Index-N) and its corresponding DNA tag adapter (DNA Index-NF_adapter and DNA Index-NR_adapter) whose length is 6bp, and the sequence directions shown in the table are all 5 '-3' direction.

Using Lasergene's PrimerSelect software, the affinity parameter between the duplexes is judged by analyzing the energy value formed between the two sequences. The larger the absolute value of the energy value (kcal/mol), the more stable the duplex result is, as follows are the energy values of the affinities of the above-mentioned DNA tag adapters, respectively, indicating that the structures formed by these DNA tag adapters are very stable.

Predicted secondary structure and energy values for DNA-tagged adapter formation. The most stable linker sequence (the most stable dimer overall).

DNA Index-1 Adapter

The most stable dimer overall: 19bp, -35.5kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCAAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTGGTT 5′

DNA Index-2 Adapter

The most stable dimer overall: 19bp, -34.0kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAACTTGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTGAAC 5′

DNA Index-3 Adapter

The most stable dimer overall: 19bp, -33.4kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGAGTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTCTCA 5′

DNA Index-4 Adapter

The most stable dimer overall: 19bp, -33.0kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAATAACT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATTATTG 5′

DNA Index-5 Adapter

The most stable dimer overall: 19bp, -36.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTACACGCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATGTGCG 5′

DNA Index-6 Adapter

The most stable dimer overall: 19bp, -34.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTACCTCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATGGAGA 5′

DNA Index-7 Adapter

The most stable dimer over rall: 19bp, -36.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTACGGAAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATGCCTT 5′

DNA Index-8 Adapter

The most stable dimer overall: 19bp, -35.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGAAGGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCTTCC 5′

DNA Index-9 Adapter

The most stable dimer overall: 19bp, -38.1kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCCGTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCGGCA 5′

DNA Index-10 Adapter

The most stable dimer overall: 19bp, -36.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCGAGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCGCTC 5′

DNA Index-11 Adapter

The most stable dimer overall: 19bp, -36.7kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGCTGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCCGAC 5′

DNA Index-12 Adapter

The most stable dimer overall: 19bp, -36.4kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTGCCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATCACGG 5′

DNA Index-13 linker

The most stable dimer overall: 19bp, -32.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTATAATCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATATTAG 5′

DNA Index-14 Adapter

The most stable dimer overall: 19bp, -33.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTATGTCAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATACAGT 5′

DNA Index-15 Adapter

The most stable dimer overall: 19bp, -35.1kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTATTCCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGATAAGGA 5′

DNA Index-16 Adapter

The most stable dimer overall: 19bp, -34.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATTCTCAACACT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTTGTG 5′

DNA Index-17 Adapter

The most stable dimer overall: 19bp, -34.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCACAAGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTGTTC 5′

DNA Index-18 Adapter

The most stable dimer overall: 19bp, -37.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCACGGTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTGCCA 5′

DNA Index-19 Adapter

The most stable dimer overall: 19bp, -34.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCACTCAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGTGAGT 5′

DNA Index-20 Adapter

The most stable dimer overall: 19bp, -37.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAACGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGTTGC 5′

DNA Index-21 Adapter

The most stable dimer overall: 19bp, -37.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAGGAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGTCCT 5′

DNA Index-22 Adapter

The most stable dimer overall: 19bp, -40.5kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGCCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGCGGA 5′

DNA Index-23 Adapter

The most stable dimer overall: 19bp, -34.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATTCTCTTACT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGGAATG 5′

DNA Index-24 Adapter

The most stable dimer overall: 19bp, -35.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAATAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCTTAT 5′

DNA Index-25 Adapter

The most stable dimer overall: 19bp, -39.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGCGTCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCGCAG 5′

DNA Index-26 Adapter

The most stable dimer overall: 19bp, -36.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGTAAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCCATT 5′

DNA Index-27 Adapter

The most stable dimer overall: 19bp, -39.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCGGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGCAGCC 5′

DNA Index-28 Adapter

The most stable dimer overall: 19bp, -34.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTAGCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGATCGA 5′

DNA Index-29 Adapter

The most stable dimer overall: 19bp, -37.5kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTCCGAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGAGGCT 5′

DNA Index-30 Adapter

The most stable dimer overall: 19bp, -34.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTTGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGACAAC 5′

DNA Index-31 Adapter

The most stable dimer overall: 19bp, -33.4kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTAGTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAGAATCA 5′

DNA Index-32 Adapter

The most stable dimer overall: 19bp, -35.5kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGACCCTCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTGGAG 5′

DNA Index-33 Adapter

The most stable dimer overall: 19bp, -37.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGCCAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTCGGT 5′

DNA Index-34 Adapter

The most stable dimer overall: 19bp, -33.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGTATT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTCATA 5′

DNA Index-35 Adapter

The most stable dimer overall: 19bp, -36.0kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGATGGAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACTACCT 5′

DNA Index-36 Adapter

The most stable dimer overall: 19bp, -35.4kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGCATAGT 3′

::: :||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACGTATC 5′

DNA Index-37 Adapter

The most stable dimer overall: 19bp, -42.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCGGCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACGGCCG 5′

DNA Index-38 Adapter

The most stable dimer overa1l: 19bp, -37.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGCGTTAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACGCAAT 5′

DNA Index-39 Adapter

The most stable dimer overall: 19bp, -36.1kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGAAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCTCTT 5′

DNA Index-40 Adapter

The most stable dimer overall: 19bp, -37.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCATGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCGTAC 5′

DNA Index-41 Adapter

The most stable dimer overall: 19bp, -40.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCGCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCGCGA 5′

DNA Index-42 Adapter

The most stable dimer overall: 19bp, -36.4kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTAGCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCATCG 5′

DNA Index-43 Adapter

The most stable dimer overall: 19bp, -37.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTTCGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACCAAGC 5′

DNA Index-44 Adapter

The most stable dimer overa1l: 19bp, -33.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTACATT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACATGTA 5′

DNA Index-45 Adapter

The most stable dimer overall: 19bp, -34.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTATCCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACATAGG 5′

DNA Index-46 Adapter

The most stable dimer overall: 19bp, -34.0kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCTGTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACAGACA 5′

DNA Index-47 Adapter

The most stable dimer overall: 19bp, -39.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGCGCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACACGCG 5′

DNA Index-48 Adapter

The most stable dimer overall: 19bp, -39.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGCGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACACCGC 5′

DNA Index-49 Adapter

The most stable dimer overall: 19bp, -33.7kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTACAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACAATGT 5′

DNA Index-50 Adapter

The most stable dimer overall: 19bp, -34.3kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTGACT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGACAACTG 5′

DNA Index-51 Adapter

The most stable dimer overall: 19bp, -35.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAATCGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATTAGC 5′

DNA Index-52 Adapter

The most stable dimer overall: 19bp, -35.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGGAGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATCCTC 5′

DNA Index-53 Adapter

The most stable dimer overall: 19bp, -35.2kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGTGCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATCACG 5′

DNA Index-54 Adapter

The most stable dimer overall: 19bp, -35.4kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTATGCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAATACGA 5′

DNA Index-55 Adapter

The most stable dimer overall: 19bp, -34.5kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAGATT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGTCTA 5′

DNA Index-56 Adapter

The most stable dimer overall: 19bp, -34.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATTCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGTAAG 5′

DNA Index-57 Adapter

The most stable dimer overall: 19bp, -37.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCAGGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGGTCC 5′

DNA Index-58 Adapter

The most stable dimer overall: 19bp, -39.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCGCAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGGCGT 5′

DNA Index-59 Adapter

The most stable dimer overall: 19bp, -34.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTACCT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGATGG 5′

DNA Index-60 Adapter

The most stable dimer overall: 19bp, -36.0kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTCGTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAGAGCA 5′

DNA Index-61 Adapter

The most stable dimer overall: 19bp, -35.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCTTAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACGAAT 5′

DNA Index-62 Adapter

The most stable dimer overall: 19bp, -36.1kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGAGAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACCTCT 5′

DNA Index-63 Adapter

The most stable dimer overall: 19bp, -35.8kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCTT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACCAGA 5′

DNA Index-64 Adapter

The most stable dimer over rall: 19bp, -33.9kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTAATT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAACATTA 5′

DNA Index-65 Adapter

The most stable dimer overall: 19bp, -34.0kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTTACTGT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAATGAC 5′

DNA Index-66 linker

The most stable dimer overall: 19bp, -33.6kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTTATAAT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAATATT 5′

DNA Index-67 Adapter

The most stable dimer overall: 19bp, -36.1kcal/mol

5′TACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCCACT 3′

::: : : ||||||||||||||||||||

3′CACTGACCTCAAGTCTGCACACGAGAAGGCTAGAAAGGTG 5′

Description of drawings

Figure 1: Schematic diagram of the routine DNA library construction process provided by Illumina.

Figure 2: Schematic diagram of the conventional DNA label library construction process provided by Illumina.

Figure 3: Schematic diagram of Illumina's DNA label library construction process.

Figure 4: Schematic flow chart of the optimized DNA tag library construction method based on adapter ligation of the present invention.

Figure 5: Electrophoresis results of the construction of 67 DNA tag libraries. (a) Electrophoresis detection results of DNA tag adapter library test (index1~index23) (lane 1 and lane 25 are D2000makrer and 50bp marker respectively, and lanes 2 to 24 are libraries constructed using DNA tag adapter index1~index23); (b ) DNA tag adapter library test (index23~index44) electrophoresis detection results (lanes 1 and 25 are D2000makrer and 50bp marker respectively, lanes 2 to 24 are libraries constructed using DNA tag adapters index23~index44, of which lane 14 is the test negative control, that is, no sample); (c) DNA tag adapter library test (index45～index67) electrophoresis detection results (lanes 1 and 25 are D2000 makrer and 50bp marker respectively, and lanes 2 to 24 use DNA tag adapter index45 respectively ~index67 constructed library).

Figure 6: The detection results of the constructed DNA tag library using Agilent2100. The sample name is Agilent3. The peaks in the figure represent Marker, sample fragment size, and Marker from left to right. The measured library fragment size is 284bp, and the concentration is 32.64ng/ul. The library size and concentration are acceptable.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention.

One aspect of the present invention provides a set of DNA tags, the DNA tags include or consist of the following: at least 5, or at least 10, of the DNA tags shown in Table 2 and which differ by 1 base, or At least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or 45, or at least 50, or at least 55, or at least 60, or all 67 indivual,

The DNA tags preferably include at least DNA Index-1 to DNA Index-5 of the 67 DNA tags shown in Table 2, or DNA Index-6 to DNA Index-10, or DNA Index-11 to DNA Index-15, or DNA Index-16～DNA Index-20, or DNA Index-21～DNA Index-25, or DNA Index-26～DNA Index-30, or DNA Index-31～DNA Index-35, or DNA Index-36～DNA Index -40, or DNA Index-41～DNA Index-45, or DNA Index-46～DNA Index-50, or DNA Index-51～DNA Index-55, or DNA Index-56～DNA Index-60, or DNA Index- 61～DNA Index-65, or DNA Index-63～DNA Index-67, or any combination of two or more of them.

In a specific embodiment of the present invention, in the DNA tag provided by the present invention, the difference of 1 base includes the substitution, addition or deletion of 1 base in the tag.

Another aspect of the present invention provides the use of the DNA tag for constructing a DNA tag library, wherein the DNA tag adapter of the DNA tag library contains the DNA tag at the 3' end, thereby constituting each corresponding DNA tag adapter, so The above-mentioned DNA tag adapter is used as the 5' adapter and the 3' adapter of the DNA tag library at the same time.

In a specific embodiment of the present invention, the use of the DNA tag for constructing a DNA tag library is provided, wherein the DNA tag is inserted into the 3' end of the DNA tag adapter, or is connected to the DNA tag adapter with or without a linker. The 3' end of the DNA adapter is preferably ligated to the 3' end of the DNA adapter without a linker.

Another aspect of the present invention provides a set of DNA tag adapters containing the DNA tags described in claim 1 or 2, wherein the DNA tag adapters of the DNA tag library contain said tags at the 3' end and simultaneously serve as 5' adapters and 3' adapters, the DNA tag adapters include or consist of the following: at least 5 of the 67 DNA tag adapters shown in Table 2 and the DNA tag sequences they contain differ by 1 base, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or 45, or at least 50, or at least 55, or at least 60 , or all 67,

The DNA tag adapter preferably includes at least DNA Index-1F/R_adapte～DNA Index-5F/R_adapter, or DNAIndex-6F/R_adapte～DNA Index-10F/R_adapter among the 67 DNA tag adapters shown in Table 2, or DNA Index-11F/R_adapte～DNA Index-15F/R_adapter, or DNA Index-16F/R_adapte～DNA Index-20F/R_adapter, or DNA Index-21F/R_adapte～DNA Index-25F/R_adapter, or DNA Index-26F/ R_adapte～DNA Index-30F/R_adapter, or DNA Index-31F/R_adapte～DNA Index-35F/R_adapter, or DNA Index-36F/R_adapte～DNA Index-40F/R_adapter, or DNA Index-41F/R_adapte～DNA Index- 45F/R_adapter, or DNA Index-46F/R_adapte～DNA Index-50F/R_adapter, or DNA Index-51F/R_adapte～DNA Index-55F/R_adapter, or DNA Index-56F/R_adapte～DNA Index-60F/R_adapter, or DNA Index-61F/R_adapte～DNA Index-65F/R_adapter, or DNA Index-66F/R_adapte～DNA Index-67F/R_adapter, or any combination of two or more of them.

In a specific embodiment of the present invention, in the DNA tag adapter, the difference of 1 base includes the substitution, addition or deletion of 1 base in the tag.

In a further aspect of the present invention, the use of the DNA tag adapter for constructing a DNA tag library is provided, preferably the DNA tag adapter is used as both the 5' adapter and the 3' adapter of the DNA tag library.

In another aspect of the present invention, a DNA tag library constructed by the DNA tag adapter provided in the present invention is provided.

Another aspect of the present invention provides a method for constructing a tag library, which is characterized in that a tag-containing DNA linker is used to construct the tag library.

In a further aspect of the present invention, a method for constructing a tag library is provided, which includes:

1) Provide n DNA samples, n is an integer and an integer of 1≤n≤67, preferably n is an integer and 2≤n≤67, the DNA samples are from all eukaryotic and prokaryotic DNA samples, including but not limited to human DNA samples;

2) Fragmenting human genomic DNA, wherein the fragmentation method includes but not limited to the ultrasonic fragmentation method, preferably the fragmented DNA bands are concentrated at about 180bp;

3) End repair;

4) Add "A" base to the 3' end of the DNA fragment;

5) connecting DNA tag adapters, wherein preferably each tag adapter is connected to both ends of the DNA fragment;

6) Gel recovery and purification of the ligated product obtained in step 5), preferably through 2% agarose gel electrophoresis and recovery, and mixing the recovered products of each DNA sample together;

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

In a specific embodiment of the present invention, in the method for constructing a tag library provided by the present invention, the DNA tag adapters include or consist of the following: the 67 DNA tag adapters shown in Table 2 and the DNA contained therein At least 5, or at least 10, or at least 15, or at least 20, at least 25, or at least 30, or at least 35, or at least 40 of the DNA tag adapters whose tag sequences differ by 1 base , or 45, or at least 50, or at least 55, or at least 60, or all 67,

The DNA tag adapter preferably includes at least DNA Index-1F/R_adapte～DNA Index-5F/R_adapter, or DNAIndex-6F/R_adapte～DNA Index-10F/R_adapter among the 67 DNA tag adapters shown in Table 2, or DNA Index-11F/R_adapte～DNA Index-15F/R_adapter, or DNA Index-16F/R_adapte～DNA Index-20F/R_adapter, or DNA Index-21F/R_adapte～DNA Index-25F/R_adapter, or DNA Index-26F/ R_adapte～DNA Index-30F/R_adapter, or DNA Index-31F/R_adapte～DNA Index-35F/R_adapter, or DNA Index-36F/R_adapte～DNA Index-40F/R_adapter, or DNA Index-41F/R_adapte～DNA Index- 45F/R_adapter, or DNA Index-46F/R_adapte～DNA Index-50F/R_adapter, or DNA Index-51F/R_adapte～DNA Index-55F/R_adapter, or DNA Index-56F/R_adapte～DNA Index-60F/R_adapter, or DNA Index-61F/R_adapte～DNA Index-65F/R_adapter, or DNA Index-66F/R_adapte～DNA Index-67F/R_adapter, or any combination of two or more of them.

In a specific embodiment of the present invention, in the method for constructing a tag library provided by the present invention, the difference of 1 base includes the substitution, addition or deletion of 1 base in the tag.

In a specific embodiment of the present invention, the present invention provides a method for constructing a tag library, wherein the primers used in step 7) PCR reaction include

PE PCR Primers 1.0:

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT; and

PE PCR Primers 2.0:

CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.

In a further aspect of the present invention, a tag library constructed by the method for constructing a tag library provided in the present invention is provided.

Example 1

Paired End DNA Oligonucleotide Sequence:

PE PCR Primers 1.0

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

PE PCR Primers 2.0

CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

Main experimental instruments and reagents

Construction of Example 1DNA Tag Library

1. Method steps

1.1 DNA interruption

5ug of human whole blood genomic DNA was interrupted for 6 minutes using a Covaris breaker (parameter settings: Duty cycle (load ratio)-20%; Intensity (strength)-5.0; Bursts persecond (pulse per second)-200; Duration ( Duration)-40seconds; Mode (mode)-Frequency sweeping (frequency sweep); Power (power)-33-34W; Temperature (temperature)-5.5to 6°C), making it the main band displayed in agarose electrophoresis Concentrate on around 180bp [5].

1.2 End Repair

Prepare the reaction mix in the following proportions:

Fragmented DNA 35μL

T4 DNA Ligase Buffer 50μL

dNTPs mixture 4μL

T4 DNA polymerase 5 μL

Klenow DNA polymerase 1 μL

T4 polynucleotide kinase 5 μL

Total volume 100μL

Adjust the comfortable constant temperature mixer to 20°C, react for 30min, then purify with the QIAquick PCR purification kit, and finally dissolve the sample in 32μL dissolution buffer.

1.3 Add "A" base to the 3' end of the DNA fragment

Prepare the reaction mixture according to the following proportions:

DNA after end repair 32μL

Klenow Enzyme Buffer 5 μL

dATP(1mM) 10μL

Klenow enzyme (3′ to 5′ exonuclease activity) 3 μL

Total volume 50μL

Adjust the comfortable thermostatic mixer to 37°C, react for 30 minutes, then purify with MiniElute PCR purification kit, and finally dissolve the sample in 10 μL Elution Buffer.

1.4 Ligation of DNA tag adapters

Prepare the reaction mixture according to the following proportions:

DNA obtained from the above steps 10μL

T4 DNA ligase buffer 25 μL

DNA Index-N adapter (synthesized by Takara Company) 10 μL

T4DNA ligase 5 μL

Total volume 50μL

Note: For different samples, use different DNA Index-N adapters (N=1～67). The DNA Index-N linker used can be the DNA index linker formed after the annealing of any pair of DNA Index-NF_adapter and DNA Index-NR_adapter shown in Table 2.

Adjust the comfortable constant temperature mixer (adjusted to 20°C, react for 15min, then use the QIAquickPCR purification kit to purify, and finally dissolve the sample in 30μL of dissolution buffer.

1.5 Gel recovery and purification of ligation products

The ligation product was separated by electrophoresis in 2% agarose gel; then the target fragment of 280-300 bp was put into an Eppendorf tube. Use the QIAquick Gel Purification Kit for gel purification and recovery, and the recovered product is dissolved in 30 μL Elution Buffer.

1.6 PCR reaction

PCR reaction: Prepare the reaction mixture according to the following reaction system, and place the reagents on ice.

Gel recovery purified DNA 10μL

PE PCR Primers 1.0 1μL

PE PCR Primers 2.0 1μL

Phusion DNA Polymerase 25μL

ddH ₂ O 13 μL

Total volume 50μL

PCR reaction conditions

9g℃30s

72℃5min

Store at 4°C

1.7 Gel recovery and purification of PCR products

The PCR product was separated by electrophoresis in 2% agarose gel, and the target fragment of 280-300 bp was cut and recovered, and the gel purification was performed with the QIAquick gel purification kit, and the recovered product was dissolved in 30 μL Elution Buffer.

1.8 DNA preparation product detection

Use the Agilent 2100 Bioanalyzer to detect the library yield according to the manufacturer's instructions (see Figure 6). The size of the measured library fragment was 284bp, and the concentration was 32.64ng/ul. The library size and concentration are acceptable.

2. Result analysis

The electrophoresis results of the 67 constructed DNA tag libraries are shown in Figure 5 below. In Figure 5, D2000 and 50bp markers were used, respectively, produced by Tiangen Company and NEB Company; the arrows marked the target library fragment size.

Statistics of Solexa sequencing results: 98.43% of tags are fully identified, ie 0 mismatches, 0.09% of tags detect 1 wrong base, 1 mismatch, and 1.48% of other reads, so sequencing Results The recognition rate of the tags was 98.5%, which can meet the sequencing requirements of the solexa DNA index.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

references

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 (44)

1. A group of DNA tags, the DNA tags at least include DNA Index-1～DNA Index-5 in the 67 DNA tags shown in Table 2. 2. A group of DNA tags according to claim 1, which also includes DNA Index-6～DNA Index-10 in the 67 DNA tags shown in Table 2. 3. A group of DNA tags according to claim 1, which also includes DNA Index-11～DNA Index-15 in the 67 DNA tags shown in Table 2. 4. A group of DNA tags as claimed in claim 1, which also includes DNA Index-16～DNA Index-20 in the 67 DNA tags shown in Table 2. 5. A set of DNA tags as claimed in claim 1, further comprising DNA Index-21～DNA Index-25 in the 67 DNA tags shown in Table 2. 6. A set of DNA tags as claimed in claim 1, further comprising DNA Index-26～DNA Index-30 in the 67 DNA tags shown in Table 2. 7. A set of DNA tags as claimed in claim 1, further comprising DNA Index-31～DNA Index-35 in the 67 DNA tags shown in Table 2. 8. A set of DNA tags as claimed in claim 1, further comprising DNA Index-36～DNA Index-40 in the 67 DNA tags shown in Table 2. 9. A set of DNA tags according to claim 1, further comprising DNA Index-41～DNA Index-45 in the 67 DNA tags shown in Table 2. 10. A set of DNA tags as claimed in claim 1, further comprising DNA Index-46～DNA Index-50 in the 67 DNA tags shown in Table 2. 11. A set of DNA tags as claimed in claim 1, further comprising DNA Index-51～DNA Index-55 in the 67 DNA tags shown in Table 2. 12. A set of DNA tags as claimed in claim 1, further comprising DNA Index-56～DNA Index-60 in the 67 DNA tags shown in Table 2. 13. A set of DNA tags as claimed in claim 1, further comprising DNA Index-61～DNA Index-65 in the 67 DNA tags shown in Table 2. 14. A set of DNA tags as claimed in claim 1, further comprising DNA Index-63～DNA Index-67 in the 67 DNA tags shown in Table 2. 15. The use of a set of DNA tags according to any one of claims 1-14 for constructing a DNA tag library, wherein the DNA tag adapters of the DNA tag library comprise the DNA tags at the 3' end, thereby forming respective corresponding DNA tag adapters. 16. The use according to claim 15, wherein the DNA tag adapter is used simultaneously as a 5' adapter and a 3' adapter of a DNA tag library. 17. The use according to claim 15, wherein the DNA tag is inserted into the 3' end of the DNA tag adapter, or connected to the 3' end of the DNA adapter with or without a linker. 18. The use according to claim 17, wherein the DNA tag is not connected to the 3' end of the DNA adapter by a linker. 19. A set of DNA tag adapters, wherein the DNA tag adapters of the DNA tag library comprise the tag of any one of claims 1-14 at the 3' end, and the DNA tag adapters include at least 67 DNAs shown in Table 2 DNA Index-1F/R_adapte～DNA Index-5F/R_adapter in index adapter. 20. A group of DNA tag adapters according to claim 19, which also includes DNA Index-6F/R_adapte～DNA Index-10F/R_adapter in the 67 DNA tag adapters shown in Table 2. 21. A set of DNA tag adapters according to claim 19, which also includes DNA Index-11F/R_adapte～DNA Index-15F/R_adapter in the 67 DNA tag adapters shown in Table 2. 22. A group of DNA tag adapters according to claim 19, which also includes DNA Index-16F/R_adapte～DNA Index-20F/R_adapter in the 67 DNA tag adapters shown in Table 2. 23. A group of DNA tag adapters according to claim 19, which also includes DNA Index-21F/R_adapte～DNA Index-25F/R_adapter in the 67 DNA tag adapters shown in Table 2. 24. A group of DNA tag adapters according to claim 19, which also includes DNA Index-26F/R_adapte～DNA Index-30F/R_adapter in the 67 DNA tag adapters shown in Table 2. 25. A group of DNA tag adapters according to claim 19, which also includes DNA Index-31F/R_adapte～DNA Index-35F/R_adapter among the 67 DNA tag adapters shown in Table 2. 26. A group of DNA tag adapters according to claim 19, which also includes DNA Index-36F/R_adapte～DNA Index-40F/R_adapter among the 67 DNA tag adapters shown in Table 2. 27. A group of DNA tag adapters according to claim 19, which also includes DNA Index-41F/R_adapte～DNA Index-45F/R_adapter among the 67 DNA tag adapters shown in Table 2. 28. A group of DNA tag adapters according to claim 19, which also includes DNA Index-46F/R_adapte～DNA Index-50F/R_adapter among the 67 DNA tag adapters shown in Table 2. 29. A set of DNA tag adapters according to claim 19, further comprising DNA Index-51F/R_adapte～DNA Index-55F/R_adapter among the 67 DNA tag adapters shown in Table 2. 30. A set of DNA tag adapters according to claim 19, further comprising DNA Index-56F/R_adapte～DNA Index-60F/R_adapter among the 67 DNA tag adapters shown in Table 2. 31. A set of DNA tag adapters according to claim 19, further comprising DNA Index-61F/R_adapte～DNA Index-65F/R_adapter among the 67 DNA tag adapters shown in Table 2. 32. A set of DNA tag adapters according to claim 19, further comprising DNA Index-66F/R_adapte～DNA Index-67F/R_adapter among the 67 DNA tag adapters shown in Table 2. 33. The set of DNA tag adapters of any one of claims 19-32, wherein the DNA tag adapters serve as both 5' and 3' adapters. 34. Use of a set of DNA tag adapters according to any one of claims 19-32 for constructing a DNA tag library. 35. The use of claim 34, wherein the DNA tagging adapter is used as both a 5' adapter and a 3' adapter of a DNA tagging library. 36. A DNA tag library constructed by a set of DNA tag adapters according to any one of claims 19-32. 37. A method for constructing a tag library, comprising: 1) Provide n DNA samples, n is an integer and an integer of 1≤n≤67, the DNA samples are from all eukaryotic and prokaryotic DNA samples, including but not limited to human DNA samples; 2) Interrupting human genomic DNA, wherein the interruption method includes but not limited to ultrasonic interruption method; 3) End repair; 4) Add "A" base to the 3' end of the DNA fragment; 5) connecting the DNA tag adapter; 6) performing gel recovery and purification on the ligated product obtained in step 5), and mixing the recovered products of each DNA sample together; 7) PCR reaction, using the mixture of recovered products in step 6) as a template, performing PCR amplification under conditions suitable for amplifying the target nucleic acid, and performing gel recovery and purification of the PCR product; Wherein the DNA tag adapter is as described in any one of claims 19-32. 38. The method of claim 37, wherein in step 1), n is an integer and 2≤n≤67. 39. The method of claim 37, wherein in step 2), the fragmented DNA bands are concentrated at about 180 bp. 40. The method of claim 37, wherein in step 5), each index adapter is ligated to both ends of the DNA fragments. 41. The method of claim 37, wherein in step 6), the ligation product is electrophoresed through 2% agarose gel and recovered. 42. The method of claim 37, wherein in step 7), the target fragment of 280-300 bp is recovered. 43. The method of claim 37, wherein step 7) primers used in the PCR reaction include PE PCR Primers1.0: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT; and PE PCR Primers 2.0: CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT. 44. A library of tags constructed by the method of claim 37.

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