CN110468188B - Tag sequence set for next generation sequencing and its design method and application - Google Patents

Abstract

The invention relates to a tag sequence set for second generation sequencing, and a design method and application thereof, belonging to the technical field of molecular biology. The length of each tag sequence in the tag sequence set is 6-10, the GC content in each tag sequence is 40-60%, the number of continuous bases in each tag sequence is 1-3 at the maximum, and the first two bases of each tag sequence are not G at the same time. The tag sequence set is used for second generation sequencing, and the number of finally obtained tag sequence sets is high through screening and combining the tag sequences, so that the requirement of large sample size detection can be met, and high-quality sequencing data can be accurately split while the sequencing requirement is met.

Description

Tag sequence set for next generation sequencing and its design method and application

technical field

The present invention relates to the field of molecular biology, in particular to a tag sequence set for next-generation sequencing and its design method and application.

Background technique

Next-generation sequencing (also known as next-generation sequencing, NGS) technology has gradually become a widely adopted technology in many aspects of discovery and translational research. Sequencing, and the method of distinguishing samples is to use the index sequence (index) to distinguish.

At present, most of the tag sequences are provided by sequencing platforms (such as Illumina), but the number of tag sequences provided by the sequencing platform is limited, and more tag sequences are required for large-scale sample sequencing or paired-end sequencing.

At present, there is a lack of a systematic method for designing tag sequences and tag sequences generated based on strict screening. The self-synthesized tag sequences may not meet the sequencing requirements, resulting in sequencing failure or difficulty in splitting the sequencing data.

Contents of the invention

Based on this, it is necessary to address the above problems and provide a tag sequence set for next-generation sequencing, which can correctly split high-quality sequencing data while meeting the sequencing requirements.

A tag sequence set for next-generation sequencing, the length of each tag sequence in the tag sequence set is 6-10, the GC content in each tag sequence is 40-60%, and the consecutive bases in each tag sequence The maximum number is 1-3, and the first two bases of each tag sequence are not G at the same time.

The tag sequences in the above tag sequence set can be used for library construction and then sequenced using a sequencing platform. It can be understood that if the designed tag sequence is used on the reverse primer, reverse complementation of the tag sequence is required.

In one embodiment, the length of the tag sequence is 8, and the maximum number of consecutive bases in each tag sequence is 2. Control the length of the tag sequence to 8, because the longer the fragment, the greater the error tolerance rate, but the long fragment also means longer sequencing time and more reagent consumption. From a practical point of view, it is more difficult to control the maximum length to 8. Good length; at the same time, the number of consecutive bases is controlled to a maximum of 2, which can maximize the balance of base content and reduce base misreading caused by sequencing.

In one embodiment, the GC content of the tag sequence is 50%. The GC content is the ratio of guanine (G) and cytosine (C) among the four bases.

In one of the embodiments, each tag sequence in the tag sequence set is pairwise compared, and for the bases at the same position, the same is recorded as 0 points, and the different ones are recorded as 1 point, and the total score is calculated. Total score ≥4 for each tag sequence. The purpose of defining the tag sequence by the integral above is to increase the Hamming distance (Hammingdistance) between the two tags, so that the two sequences can be better distinguished. For example, when the two sequences are: TGGTTACC, CAACCAGA, only the 6th base is the same as "A", so the total score of the label is 7, and another example is two sequences: CGATCACA, CAATCAGA, the total score of the label is 2 , it can be seen that the difference of the former group is greater than that of the latter group, which can better distinguish the sequences, and the recognition errors due to mismatches are greatly reduced.

In one of the embodiments, the total score of each tag sequence is 4. The method has the following advantages: 1. To ensure the maximum diversity of tag sequences; 2. To obtain enough tag sequences for production.

In one embodiment, the tag sequence is selected from the nucleotide sequences shown in SEQ ID No.1-SEQ ID No.142. The above-mentioned tag sequence can correctly split high-quality sequencing data while meeting the sequencing requirements. The details are shown in the following table:

Table 1. Tag sequence

In one embodiment, the base content ratios of the sequencing tags at the same position are: A=20-30%, B=20-30%, C=20-30%, D=20-30%. Controlling the base content at the same position within the above range has the following advantages: it can not only ensure the base balance, avoid the light diffraction caused by some signals at the same position that are too high, but also avoid some signals at the same position that are too low and miss reading.

The present invention also discloses the application of the above-mentioned tag sequence set for next-generation sequencing in the sequencing of DNA and/or RNA PCR products of pathogenic microorganisms.

The present invention also discloses the above-mentioned method for designing a tag sequence set for next-generation sequencing, including the following steps:

1) List the sequences of all base combinations of the predetermined tag length;

2) Selecting sequences that meet the predetermined GC content;

3) Screen the sequence with the maximum number of consecutive bases in the above sequence being 2;

4) Screen out the sequences whose first two bases are different from G in the above sequence;

5) Compare the above sequences pairwise. For the bases at the same position, the same base is scored as 0 points, and the different bases are scored as 1 point. The total score is calculated, and the sequence with a total score ≥ 4 is selected;

6) According to the number of required tag sequences, select sequences from the above sequences, so that the base content ratio of the obtained sequencing tags at the same position is: A=20-30%, B=20-30%, C=20-30% , D = 20-30%.

Compared with the prior art, the present invention has the following beneficial effects:

A tag sequence set for next-generation sequencing of the present invention, through the screening and combination of tag sequences, the number of tag sequence sets finally obtained is relatively high, which can meet the requirements of large sample size detection, and can meet the sequencing requirements at the same time It can correctly split high-quality sequencing data.

Detailed ways

In order to facilitate understanding of the present invention, the present invention will be described more fully below with reference to Examples. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example 1

Using the tag sequence set of the present invention to carry out next-generation sequencing, the steps are as follows:

1. Synthesize adapter primers.

Adapter primers were synthesized according to the table below and diluted to 10 μM.

Table 2. List of primer sequences

Note: The underlined part in the sequence in the above table indicates the tag sequence, in which SEQ ID No.146-148 uses the reverse complementary sequence.

2. Sample extraction.

According to the method of Qiagen QIAamp Viral RNA Mini Kit (Cat. No.: 52906), RNA of different sample types was extracted, and the concentration of RNA samples was accurately quantified with a Qubit 3.0 fluorescence quantitator. The results are shown in the table below.

Table 3. Extraction and selection index of each sample

3. Library construction and on-machine sequencing.

VAHTS RNA library construction kit (NR603) was used for RNA library construction and detection on Illumina NEXTSEQ550. The quality control results of the machine data obtained are shown in the table below.

Table 4. Quality control results table of machine data results

sample number Total reads Clean reads Adapter ratio(%) Q20(%) Q30(%) M474 15729086 15340309 2.47 95.72 93.82 M483 16292591 15590119 4.31 95.51 93.54 M522 33455397 33418373 0.11 96.76 95.17

From the above results, it can be seen that the Q30 of the data is > 93%, and the Adapter ratio is less than 5%, indicating that the requirements for library construction and computer use are still met under the condition of extremely low initial amount.

Example 2 (double index)

Using the tag sequence set of the present invention to carry out next-generation sequencing is basically the same as in Example 1, except that the tag sequences used are shown in the table below.

Table 5. List of primer sequences

Note: The underlined part in the sequence in the above table indicates the tag sequence, in which SEQ ID No.149-180 uses the reverse complementary sequence.

Sequencing method:

1. Synthesize the linker primers in Table 5 and dilute to 10 μM.

2. Sample extraction.

According to the method of Genomic DNA Extraction Kit (DP316) from Beijing Tiangen Biochemical Co., Ltd., the DNA of cerebrospinal fluid was extracted, and the concentration of RNA samples was accurately quantified by Qubit 3.0 fluorescence quantification instrument. The results are shown in the table below.

Table 6. Extraction and selection index of each sample

3. Library construction and on-machine sequencing.

The VAHTS library construction kit IndexuePrep DNA Library Prep Kit V2 for Illumina (TD503-01) was used for DNA library construction and sequenced on Illumina NEXTSEQ550. The quality control results of the machine data obtained are shown in the table below.

Table 7. Quality control results

sample number Total reads Clean reads Adapter ratio(%) Q20(%) Q30(%) TM113 11,452,816 11,377,949 0.65 97.31 96.08 TM114 11,939,764 11,838,146 0.85 97.37 96.14 TM115 15,922,031 15,770,616 0.95 97.3 96.06 TM116 10,488,031 10,423,284 0.62 97.44 96.25 TM213 13,888,422 13,835,666 0.38 97.7 96.65 TM214 10,528,662 10,509,329 0.18 97.45 96.29 TM215 9,891,360 9,875,685 0.16 96.44 94.58 TM216 12,910,386 12,845,906 0.5 97.53 96.41 TM313 12,297,899 12,221,931 0.62 96.72 95.13 TM314 11,923,854 11,858,064 0.55 97.55 96.43 TM315 11,674,479 11,625,816 0.42 97.22 95.91 TM316 11,751,190 11,692,215 0.5 97.54 96.39 TM413 11,591,677 11,528,769 0.54 96.01 94.04 TM414 12,954,704 12,903,730 0.39 97.65 96.59 TM415 9,792,747 9,765,468 0.28 96.41 94.61 TM416 10,679,321 10,638,169 0.39 97.29 96.03 TM513 12,730,902 12,668,132 0.49 97.4 96.17 TM514 10,705,553 10,671,839 0.31 97.66 96.59 TM515 11,601,832 11,544,594 0.49 97.36 96.14 TM516 11,656,420 11,604,493 0.45 97.7 96.65 TM613 11,945,478 11,895,750 0.42 97.54 96.4 TM614 12,593,749 12,538,150 0.44 97.26 95.95 TM615 12,368,344 12,179,404 1.53 97.66 96.57 TM616 10,984,521 10,910,528 0.67 97.55 96.42 TM713 14,895,002 14,827,596 0.45 97.33 96.05 TM714 11,387,099 11,332,418 0.48 97.39 96.19 TM715 11,193,700 11,115,502 0.7 97.45 96.24 TM716 10,350,481 10,292,859 0.56 97.38 96.14 TM813 9,882,766 9,843,323 0.4 97.24 95.94 TM814 9,236,615 9,202,806 0.37 97.09 95.71 TM815 10,289,606 10,243,417 0.45 97.04 95.64 TM816 12,072,236 11,928,800 1.19 97.38 96.16

Table 8. Data split size

From the above results, it can be seen that the Q30 of the data is >95%, the Adaptor ratio is less than 2%, and the data extracted from i5 and i7 are balanced, indicating that the designed linker sequence can meet the requirements of large-scale library construction.

To sum up, the label sequence set of the present invention can overcome the problems of the small number of existing labels, the difficulty in realizing the simultaneous detection of a large sample size, and the low error tolerance rate, which easily leads to data splitting errors, resulting in false positives or false negatives. In addition, some tag sequences have too long continuous sequences, which are difficult to interpret during the sequencing process, and the GC base balance is poor, which easily leads to poor data quality and other problems. At the same time, by designing an appropriate Hamming distance, the error rate of the machine reading bases is reduced. It not only has the advantages of a large number of tag sequence sets, which can meet the requirements of large sample size detection, but also can correctly split high-quality sequencing data while meeting the sequencing requirements.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

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<211> 8

<212>DNA

<213> Artificial Sequence

<400> 118

accatcct 8

<210> 119

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 119

tgactcct8

<210> 120

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 120

cctgtaac 8

<210> 121

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 121

acctaacc 8

<210> 122

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 122

tgtactcg 8

<210> 123

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 123

cgatatcc 8

<210> 124

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 124

acctgtgt8

<210> 125

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 125

tgttgagg 8

<210> 126

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 126

cgagt 8

<210> 127

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 127

acgattgg 8

<210> 128

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 128

tgtgagac 8

<210> 129

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 129

cgtaacca 8

<210> 130

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 130

acgacaca 8

<210> 131

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 131

tgtgtctg8

<210> 132

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 132

cgtacgtt 8

<210> 133

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 133

agacacag 8

<210> 134

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 134

tgctctga 8

<210> 135

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 135

cgtggata 8

<210> 136

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 136

agactgtc 8

<210> 137

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 137

tggaagct 8

<210> 138

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 138

agagcaac 8

<210> 139

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 139

tggatcga 8

<210> 140

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 140

agaggttg 8

<210> 141

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 141

tggttacc 8

<210> 142

<211> 8

<212>DNA

<213> Artificial Sequence

<400> 142

agcaactc 8

<210> 143

<211> 70

<212>DNA

<213> Artificial Sequence

<400> 143

aatgatacgg cgaccaccga gatctacact gtactcgaca ctctttccct acacgacgct 60

cttccgatct70

<210> 144

<211> 70

<212>DNA

<213> Artificial Sequence

<400> 144

aatgatacgg cgaccaccga gatctacacc attcctgaca ctctttccct acacgacgct 60

cttccgatct70

<210> 145

<211> 70

<212>DNA

<213> Artificial Sequence

<400> 145

aatgatacgg cgaccaccga gatctacacc aggaatcaca ctctttccct acacgacgct 60

cttccgatct70

<210> 146

<211> 66

<212>DNA

<213> Artificial Sequence

<400> 146

caagcagaag acggcatacg agatcgagac ttgtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 147

<211> 66

<212>DNA

<213> Artificial Sequence

<400> 147

caagcagaag acggcatacg agattccttg gtgtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 148

<211> 66

<212>DNA

<213> Artificial Sequence

<400> 148

caagcagaag acggcatacg agatactctc gagtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 149

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 149

caagcagaag acggcatacg agatgtctgg atgtctcgtg ggctcgg 47

<210> 150

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 150

caagcagaag acggcatacg agatgctgat gagtctcgtg ggctcgg 47

<210> 151

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 151

caagcagaag acggcatacg agattgcagt aggtctcgtg ggctcgg 47

<210> 152

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 152

caagcagaag acggcatacg agatcgtatt gcgtctcgtg ggctcgg 47

<210> 153

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 153

caagcagaag acggcatacg agatcagagg atgtctcgtg ggctcgg 47

<210> 154

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 154

caagcagaag acggcatacg agatcgacat gagtctcgtg ggctcgg 47

<210> 155

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 155

caagcagaag acggcatacg agatctctct aggtctcgtg ggctcgg 47

<210> 156

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 156

caagcagaag acggcatacg agatacagtt gcgtctcgtg ggctcgg 47

<210> 157

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 157

caagcagaag acggcatacg agatgagtgt gagtctcgtg ggctcgg 47

<210> 158

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 158

caagcagaag acggcatacg agatgctacg atgtctcgtg ggctcgg 47

<210> 159

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 159

caagcagaag acggcatacg agatctcagt gagtctcgtg ggctcgg 47

<210> 160

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 160

caagcagaag acggcatacg agatcttgga aggtctcgtg ggctcgg 47

<210> 161

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 161

caagcagaag acggcatacg agattaacact gcgtctcgtg ggctcgg 47

<210> 162

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 162

caagcagaag acggcatacg agatcaggtt gtgtctcgtg ggctcgg 47

<210> 163

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 163

caagcagaag acggcatacg agatgaacga aggtctcgtg ggctcgg 47

<210> 164

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 164

caagcagaag acggcatacg agatggatta gcgtctcgtg ggctcgg 47

<210> 165

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 165

caagcagaag acggcatacg agatgtcctt gtgtctcgtg ggctcgg 47

<210> 166

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 166

caagcagaag acggcatacg agatcaccta gagtctcgtg ggctcgg 47

<210> 167

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 167

caagcagaag acggcatacg agattgacag aggtctcgtg ggctcgg 47

<210> 168

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 168

caagcagaag acggcatacg agatagctgt gtgtctcgtg ggctcgg 47

<210> 169

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 169

caagcagaag acggcatacg agatcgttga gagtctcgtg ggctcgg 47

<210> 170

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 170

caagcagaag acggcatacg agattcgagt gtgtctcgtg ggctcgg 47

<210> 171

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 171

caagcagaag acggcatacg agatgcaaga gagtctcgtg ggctcgg 47

<210> 172

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 172

caagcagaag acggcatacg agatcgttac aggtctcgtg ggctcgg 47

<210> 173

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 173

caagcagaag acggcatacg agataaggag gagtctcgtg ggctcgg 47

<210> 174

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 174

caagcagaag acggcatacg agatggaact gtgtctcgtg ggctcgg 47

<210> 175

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 175

caagcagaag acggcatacg agattccag gagtctcgtg ggctcgg 47

<210> 176

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 176

caagcagaag acggcatacg agattcatcc aggtctcgtg ggctcgg 47

<210> 177

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 177

caagcagaag acggcatacg agatctcgaa gtgtctcgtg ggctcgg 47

<210> 178

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 178

caagcagaag acggcatacg agatgagcaa gtgtctcgtg ggctcgg 47

<210> 179

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 179

caagcagaag acggcatacg agataaccat gggtctcgtg ggctcgg 47

<210> 180

<211> 47

<212>DNA

<213> Artificial Sequence

<400> 180

caagcagaag acggcatacg agattcagca gtgtctcgtg ggctcgg 47

<210> 181

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 181

aatgatacgg cgaccaccga gatctacaca atccacctcg tcggcagcgt c 51

<210> 182

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 182

aatgatacgg cgaccaccga gatctacact aagcacgtcg tcggcagcgt c 51

<210> 183

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 183

aatgatacgg cgaccaccga gatctacacc aaccagatcg tcggcagcgt c 51

<210> 184

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 184

aatgatacgg cgaccaccga gatctacaca gcatgagtcg tcggcagcgt c 51

<210> 185

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 185

aatgatacgg cgaccaccga gatctacacg aacctagtcg tcggcagcgt c 51

<210> 186

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 186

aatgatacgg cgaccaccga gatctacaca atcgtggtcg tcggcagcgt c 51

<210> 187

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 187

aatgatacgg cgaccaccga gatctacact aaggtgctcg tcggcagcgt c 51

<210> 188

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 188

aatgatacgg cgaccaccga gatctacaca ggtcctatcg tcggcagcgt c 51

<210> 189

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 189

aatgatacgg cgaccaccga gatctacacc aacgtcttcg tcggcagcgt c 51

<210> 190

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 190

aatgatacgg cgaccaccga gatctacaca acacagggtcg tcggcagcgt c 51

<210> 191

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 191

aatgatacgg cgaccaccga gatctacact accaagctcg tcggcagcgt c 51

<210> 192

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 192

aatgatacgg cgaccaccga gatctacaca ggtggattcg tcggcagcgt c 51

<210> 193

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 193

aatgatacgg cgaccaccga gatctacacc atactgctcg tcggcagcgt c 51

<210> 194

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 194

aatgatacgg cgaccaccga gatctacacg atgcagttcg tcggcagcgt c 51

<210> 195

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 195

aatgatacgg cgaccaccga gatctacaca acagtcctcg tcggcagcgt c 51

<210> 196

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 196

aatgatacgg cgaccaccga gatctacact accttcgtcg tcggcagcgt c 51

<210> 197

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 197

aatgatacgg cgaccaccga gatctacacc atagacgtcg tcggcagcgt c 51

<210> 198

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 198

aatgatacgg cgaccaccga gatctacacg atggtcatcg tcggcagcgt c 51

<210> 199

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 199

aatgatacgg cgaccaccga gatctacaca actccactcg tcggcagcgt c 51

<210> 200

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 200

aatgatacgg cgaccaccga gatctacacg agtaggatcg tcggcagcgt c 51

<210> 201

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 201

aatgatacgg cgaccaccga gatctacaca actggtgtcg tcggcagcgt c 51

<210> 202

<211> 51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 202

aatgatacgg cgaccaccga gatctacacc attggactcg tcggcagcgt c 51

<210> 203

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 203

aatgatacgg cgaccaccga gatctacacg agttccttcg tcggcagcgt c 51

<210> 204

<211> 51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 204

aatgatacgg cgaccaccga gatctacaca accaccatcg tcggcagcgt c 51

<210> 205

<211> 51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 205

aatgatacgg cgaccaccga gatctacact agcagtgtcg tcggcagcgt c 51

<210> 206

<211> 51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 206

aatgatacgg cgaccaccga gatctacacc atgagcttcg tcggcagcgt c 51

<210> 207

<211> 51

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 207

aatgatacgg cgaccaccga gatctacacg tatgctgtcg tcggcagcgt c 51

<210> 208

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 208

aatgatacgg cgaccaccga gatctacaca acctggttcg tcggcagcgt c 51

<210> 209

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 209

aatgatacgg cgaccaccga gatctacact agctcactcg tcggcagcgt c 51

<210> 210

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 210

aatgatacgg cgaccaccga gatctacacg tagaacctcg tcggcagcgt c 51

<210> 211

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 211

aatgatacgg cgaccaccga gatctacact tcacacctcg tcggcagcgt c 51

<210> 212

<211> 51

<212>DNA

<213> Artificial Sequence

<400> 212

aatgatacgg cgaccaccga gatctacacc acacgaatcg tcggcagcgt c 51

Claims (1)

1. A tag sequence set for next-generation sequencing, characterized in that it includes the nucleotide sequences shown in SEQ ID No. 1-SEQ ID No. 142 and their reverse complementary sequences.