CN110468188B - Tag sequence set for second generation sequencing and design method and application thereof - Google Patents
Tag sequence set for second generation sequencing and design method and application thereof Download PDFInfo
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- CN110468188B CN110468188B CN201910779115.8A CN201910779115A CN110468188B CN 110468188 B CN110468188 B CN 110468188B CN 201910779115 A CN201910779115 A CN 201910779115A CN 110468188 B CN110468188 B CN 110468188B
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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
Technical Field
The invention relates to the field of molecular biology, in particular to a tag sequence set for second generation sequencing, and a design method and application thereof.
Background
Second generation sequencing (also called next generation sequencing, NGS) technology has become a widely adopted technology in many aspects of discovery and transformation research, and due to the high-throughput characteristic, second generation sequencing can simultaneously sequence a plurality of samples, and a method for distinguishing samples is to use tag sequences (index) for distinguishing.
At present, most of tag sequences are sequences provided by a sequencing platform (such as Illumina), but the number of tag sequences provided by the sequencing platform is limited, and more tag sequences are required when large-scale sample sequencing or double-ended sequencing is performed.
At present, a method for designing a tag sequence by independently designing the tag sequence lacks a system and the tag sequence generated based on strict screening, and the tag sequence synthesized by the method is possibly not in accordance with the sequencing requirement, so that the sequencing is failed or the sequencing data is difficult to split.
Disclosure of Invention
In view of the foregoing, it is desirable to provide a set of tag sequences for use in second generation sequencing that are capable of correctly resolving high quality sequencing data while meeting sequencing requirements.
A set of tag sequences for use in second generation sequencing, each tag sequence in the set having a length of from 6 to 10, a GC content in each tag sequence of from 40 to 60%, a number of consecutive bases in each tag sequence of from 1 to 3 at the maximum, and the first two bases of each tag sequence being not simultaneously G.
The tag sequences in the tag sequence set described above can be used in library construction and subsequently sequenced using a sequencing platform. It will be appreciated that reverse complementarity of the tag sequence is required as the designed tag sequence is used on the reverse primer.
In one embodiment, the tag sequences have a length of 8, and the number of consecutive bases in each tag sequence is at most 2. The length of the tag sequence is controlled to be 8, and the longer the fragment is, the greater the fault tolerance rate is, but the longer fragment also means longer sequencing time and more reagent consumption, and from the practical point of view, the maximum length is controlled to be 8 to be a better length; meanwhile, the maximum number of continuous bases is controlled to be 2, so that the base misreading caused by sequencing can be reduced while the balance of the base content is ensured to the maximum extent.
In one embodiment, the GC content of the tag sequence is 50%. GC content is the ratio of guanine (G) to cytosine (C) in the 4 bases.
In one embodiment, each tag sequence in the set of tag sequences is aligned pairwise, and for bases at the same position, the same score is 0, and the different score is 1, and the total score is calculated, wherein the total score of each tag sequence is equal to or greater than 4. The objective of the above-described integration of tag sequences is to increase the Hamming distance between two tags, so that the two sequences can be better distinguished. For example, when two sequences are: TGGTTACC, CAACCAGA, only the 6 th base is the same as "A", so the tag is always divided into 7, and the tag is always divided into 2 if two sequences are CGATCACA, CAATCAGA, so that the difference of the former group is larger than that of the latter group, the sequences can be better distinguished, and the error of recognition caused by mismatch is greatly reduced.
In one embodiment, the total score of each tag sequence is 4. Has the following advantages: 1, guaranteeing the difference of the tag sequences to the maximum extent; 2, a tag sequence sufficient for production can be obtained.
In one embodiment, the tag sequence is selected from the nucleotide sequences set forth in SEQ ID No.1-SEQ ID No. 142. The tag sequence can meet the sequencing requirement and can accurately separate high-quality sequencing data. The specific table is shown below:
TABLE 1 tag sequences
In one embodiment, the base content ratio of the sequencing tag at the same position is: a=20-30%, b=20-30%, c=20-30%, d=20-30%. The control of the base content at the same position within the above range has the following advantages: the base balance can be ensured, and the light diffraction generated by overhigh signals at the same position can be avoided; but also can avoid that some signals at the same position are too low to be read.
The invention also discloses application of the tag sequence set for second generation sequencing in sequencing DNA and/or RNA PCR products of pathogenic microorganisms.
The invention also discloses a design method of the tag sequence set for the second generation sequencing, which comprises the following steps:
1) Listing the sequences of all base combinations of a predetermined tag length;
2) Selecting sequences wherein the sequences meet a predetermined GC content;
3) Screening sequences with the number of continuous bases of 2 at most from the sequences;
4) Screening the sequences with G at the same time when the first two bases are different from each other;
5) Comparing the sequences in pairs, marking the same base as 0 score and the different base as 1 score for the same base, calculating total score, and selecting the sequence with the total score not less than 4;
6) Selecting sequences from the sequences according to the number of the required tag sequences, so that the base content ratio of the obtained sequencing tag at the same position is as follows: a=20-30%, b=20-30%, c=20-30%, d=20-30%.
Compared with the prior art, the invention has the following beneficial effects:
according to the tag sequence set for second generation sequencing, the tag sequences are screened and combined, so that the number of finally obtained tag sequence sets is high, 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.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the embodiments that are illustrated below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
The tag sequence set of the invention is adopted for second generation sequencing, and the steps are as follows:
1. the adaptor primer was synthesized.
The adaptor primers were synthesized according to the following table and diluted to 10. Mu.M.
TABLE 2 primer sequence listing
Note that: the underlined parts of the sequences in the above table represent the tag sequences, with SEQ ID Nos. 146-148 using the reverse complement sequences.
2. And (5) sample extraction.
RNA of different sample types was extracted according to the method of Qiagen QIAamp Viral RNA Mini Kit (cat: 52906), and the concentration of the RNA sample was accurately quantified by using a Qubit 3.0 fluorescence quantification apparatus. The results are shown in the following table.
TABLE 3 extraction and selection of index for each sample
3. Library construction and on-press sequencing.
RNA library construction was performed using the VAHTS RNA library kit (NR 603) and then tested on-line on an Illumina NEXTSEQ 550. The quality control of the obtained on-machine data is shown in the following table.
TABLE 4 quality control results table for on-line data results
| Sample numbering | 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 of warehouse building and on-boarding are still met at very low initial amounts.
Example 2 (double index)
Second generation sequencing was performed using the tag sequence set of the present invention, substantially the same as in example 1, except that the tag sequences used are as shown in the following table.
TABLE 5 primer sequence listing
Note that: the underlined parts of the sequences in the above table represent the tag sequences, wherein SEQ ID Nos. 149-180 use the reverse complement sequences.
Sequencing method:
1. the adaptor primers in Table 5 were synthesized and diluted to 10. Mu.M.
2. And (5) sample extraction.
Extracting DNA of cerebrospinal fluid according to a method of extracting a microsample genome DNA kit (DP 316) of Beijing Tiangen Biochemical Co., ltd.) and accurately quantifying the concentration of an RNA sample by using a Qubit 3.0 fluorescence quantitative instrument. The results are shown in the following table.
TABLE 6 extraction and selection of index for each sample
3. Library construction and on-press sequencing.
DNA library construction was performed using the VAHTS library kit IndexuePrep DNA Library Prep Kit V for Illumina (TD 503-01) followed by machine sequencing on Illumina NEXTSEQ 550. The quality control of the obtained on-machine data is shown in the following table.
TABLE 7 quality control results
| Sample numbering | 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 results, the Q30 of the data is more than 95%, the ratio of the adapter is less than 2%, and the data obtained by disassembling i5 and i7 are balanced, so that the designed joint sequence can meet the requirement of large-scale database construction.
In summary, the tag sequence set of the invention can overcome the problems that the number of the existing tags is small, the simultaneous detection of a large sample size is difficult to realize, the fault tolerance rate is low, the data splitting error is easy to cause, and the false positive or the false negative is generated. And part of the tag sequence has the problems of overlong continuous sequence, difficult interpretation in the sequencing process, poor base balance of GC, easiness in causing poor data quality and the like. Meanwhile, by designing a proper Hamming distance, the error rate of the machine for reading the base is reduced. The method has the advantages of large number of tag sequence sets, capability of realizing the requirement of large sample size detection, and capability of accurately separating out high-quality sequencing data while meeting the sequencing requirement.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Guangzhou microphoton Gene technology Co., ltd
<120> tag sequence set for second generation sequencing, design method and application thereof
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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catagacg 8
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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aactccac 8
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<213> Artificial sequence (Artificial Sequence)
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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cattcctg 8
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gagtagga 8
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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tacgtgtc 8
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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<211> 8
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<211> 8
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<210> 34
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catgtcga 8
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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gtagaacc 8
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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aagacgtc 8
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ttcacacc 8
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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cacagctt 8
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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aagtctcg 8
<210> 45
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
ttcgaggt 8
<210> 46
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
cactatgg 8
<210> 47
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
gttcgaga 8
<210> 48
<211> 8
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<213> Artificial sequence (Artificial Sequence)
<400> 48
aagtgagc 8
<210> 49
<211> 8
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<213> Artificial sequence (Artificial Sequence)
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<210> 50
<211> 8
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<400> 50
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<210> 51
<211> 8
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<213> Artificial sequence (Artificial Sequence)
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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<210> 60
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atacctgc 8
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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gtgttgtc 8
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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<211> 8
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<213> Artificial sequence (Artificial Sequence)
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ttgctagg 8
<210> 66
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ctacacgt 8
<210> 67
<211> 8
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<213> Artificial sequence (Artificial Sequence)
<400> 67
gtgcacaa 8
<210> 68
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
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<210> 69
<211> 8
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<213> Artificial sequence (Artificial Sequence)
<400> 69
tcatcagc 8
<210> 70
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
ctactgca 8
<210> 71
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
gcaatacg 8
<210> 72
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
atcctctg 8
<210> 73
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
tcatgtcg 8
<210> 74
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
ctagagag 8
<210> 75
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
gcaactgt 8
<210> 76
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
atcgatcg 8
<210> 77
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
tcacactc 8
<210> 78
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ctagtctc 8
<210> 79
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gcagacat 8
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<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atcgtagc 8
<210> 81
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
tcactgag 8
<210> 82
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
cttccaag 8
<210> 83
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
gcagtgta 8
<210> 84
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
acaacctg 8
<210> 85
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
tctaccac 8
<210> 86
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 86
cttcgttc 8
<210> 87
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
gctaatcc 8
<210> 88
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 88
acaaggac 8
<210> 89
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
tctaggtg 8
<210> 90
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
ctctgtca 8
<210> 91
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
acacagct 8
<210> 92
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
tctcaacg 8
<210> 93
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
ctgatggt 8
<210> 94
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
acactcga 8
<210> 95
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
tctcttgc 8
<210> 96
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
ctgtaacg 8
<210> 97
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
acagaagg 8
<210> 98
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
tcctcctt 8
<210> 99
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
ctgtctac 8
<210> 100
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
acagttcc 8
<210> 101
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
tcctggaa 8
<210> 102
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 102
ctggatga 8
<210> 103
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 103
acttcgag 8
<210> 104
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 104
tcgagagt 8
<210> 105
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 105
ccatccaa 8
<210> 106
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
acttgctc 8
<210> 107
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
tcgtacca 8
<210> 108
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
ccatggtt 8
<210> 109
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 109
actgctga 8
<210> 110
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 110
tgatcgtg 8
<210> 111
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 111
cctcagaa 8
<210> 112
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
actggact 8
<210> 113
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
tgatgcac 8
<210> 114
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
cctctctt 8
<210> 115
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 115
accaagga 8
<210> 116
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 116
tgacagga 8
<210> 117
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 117
cctgattg 8
<210> 118
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 118
accatcct 8
<210> 119
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 119
tgactcct 8
<210> 120
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 120
cctgtaac 8
<210> 121
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 121
acctaacc 8
<210> 122
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 122
tgtactcg 8
<210> 123
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 123
cgatatcc 8
<210> 124
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 124
acctgtgt 8
<210> 125
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 125
tgttgagg 8
<210> 126
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 126
cgagtagt 8
<210> 127
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 127
acgattgg 8
<210> 128
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 128
tgtgagac 8
<210> 129
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 129
cgtaacca 8
<210> 130
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 130
acgacaca 8
<210> 131
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 131
tgtgtctg 8
<210> 132
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 132
cgtacgtt 8
<210> 133
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 133
agacacag 8
<210> 134
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 134
tgctctga 8
<210> 135
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 135
cgtggata 8
<210> 136
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 136
agactgtc 8
<210> 137
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 137
tggaagct 8
<210> 138
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 138
agagcaac 8
<210> 139
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 139
tggatcga 8
<210> 140
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 140
agaggttg 8
<210> 141
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 141
tggttacc 8
<210> 142
<211> 8
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 142
agcaactc 8
<210> 143
<211> 70
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 143
aatgatacgg cgaccaccga gatctacact gtactcgaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 144
<211> 70
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 144
aatgatacgg cgaccaccga gatctacacc attcctgaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 145
<211> 70
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 145
aatgatacgg cgaccaccga gatctacacc aggaatcaca ctctttccct acacgacgct 60
cttccgatct 70
<210> 146
<211> 66
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 146
caagcagaag acggcatacg agatcgagac ttgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 147
<211> 66
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 147
caagcagaag acggcatacg agattccttg gtgtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 148
<211> 66
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 148
caagcagaag acggcatacg agatactctc gagtgactgg agttcagacg tgtgctcttc 60
cgatct 66
<210> 149
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 149
caagcagaag acggcatacg agatgtctgg atgtctcgtg ggctcgg 47
<210> 150
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 150
caagcagaag acggcatacg agatgctgat gagtctcgtg ggctcgg 47
<210> 151
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 151
caagcagaag acggcatacg agattgcagt aggtctcgtg ggctcgg 47
<210> 152
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 152
caagcagaag acggcatacg agatcgtatt gcgtctcgtg ggctcgg 47
<210> 153
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 153
caagcagaag acggcatacg agatcagagg atgtctcgtg ggctcgg 47
<210> 154
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 154
caagcagaag acggcatacg agatcgacat gagtctcgtg ggctcgg 47
<210> 155
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 155
caagcagaag acggcatacg agatctctct aggtctcgtg ggctcgg 47
<210> 156
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 156
caagcagaag acggcatacg agatacagtt gcgtctcgtg ggctcgg 47
<210> 157
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 157
caagcagaag acggcatacg agatgagtgt gagtctcgtg ggctcgg 47
<210> 158
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 158
caagcagaag acggcatacg agatgctacg atgtctcgtg ggctcgg 47
<210> 159
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 159
caagcagaag acggcatacg agatctcagt gagtctcgtg ggctcgg 47
<210> 160
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 160
caagcagaag acggcatacg agatcttgga aggtctcgtg ggctcgg 47
<210> 161
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 161
caagcagaag acggcatacg agattacact gcgtctcgtg ggctcgg 47
<210> 162
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 162
caagcagaag acggcatacg agatcaggtt gtgtctcgtg ggctcgg 47
<210> 163
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 163
caagcagaag acggcatacg agatgaacga aggtctcgtg ggctcgg 47
<210> 164
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 164
caagcagaag acggcatacg agatggatta gcgtctcgtg ggctcgg 47
<210> 165
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 165
caagcagaag acggcatacg agatgtcctt gtgtctcgtg ggctcgg 47
<210> 166
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 166
caagcagaag acggcatacg agatcaccta gagtctcgtg ggctcgg 47
<210> 167
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 167
caagcagaag acggcatacg agattgacag aggtctcgtg ggctcgg 47
<210> 168
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 168
caagcagaag acggcatacg agatagctgt gtgtctcgtg ggctcgg 47
<210> 169
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 169
caagcagaag acggcatacg agatcgttga gagtctcgtg ggctcgg 47
<210> 170
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 170
caagcagaag acggcatacg agattcgagt gtgtctcgtg ggctcgg 47
<210> 171
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 171
caagcagaag acggcatacg agatgcaaga gagtctcgtg ggctcgg 47
<210> 172
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 172
caagcagaag acggcatacg agatcgttac aggtctcgtg ggctcgg 47
<210> 173
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 173
caagcagaag acggcatacg agataaggag gagtctcgtg ggctcgg 47
<210> 174
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 174
caagcagaag acggcatacg agatggaact gtgtctcgtg ggctcgg 47
<210> 175
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 175
caagcagaag acggcatacg agatttccag gagtctcgtg ggctcgg 47
<210> 176
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 176
caagcagaag acggcatacg agattcatcc aggtctcgtg ggctcgg 47
<210> 177
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 177
caagcagaag acggcatacg agatctcgaa gtgtctcgtg ggctcgg 47
<210> 178
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 178
caagcagaag acggcatacg agatgagcaa gtgtctcgtg ggctcgg 47
<210> 179
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 179
caagcagaag acggcatacg agataaccat gggtctcgtg ggctcgg 47
<210> 180
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 180
caagcagaag acggcatacg agattcagca gtgtctcgtg ggctcgg 47
<210> 181
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 181
aatgatacgg cgaccaccga gatctacaca atccacctcg tcggcagcgt c 51
<210> 182
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 182
aatgatacgg cgaccaccga gatctacact aagcacgtcg tcggcagcgt c 51
<210> 183
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 183
aatgatacgg cgaccaccga gatctacacc aaccagatcg tcggcagcgt c 51
<210> 184
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 184
aatgatacgg cgaccaccga gatctacaca gcatgagtcg tcggcagcgt c 51
<210> 185
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 185
aatgatacgg cgaccaccga gatctacacg aacctagtcg tcggcagcgt c 51
<210> 186
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 186
aatgatacgg cgaccaccga gatctacaca atcgtggtcg tcggcagcgt c 51
<210> 187
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 187
aatgatacgg cgaccaccga gatctacact aaggtgctcg tcggcagcgt c 51
<210> 188
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 188
aatgatacgg cgaccaccga gatctacaca ggtcctatcg tcggcagcgt c 51
<210> 189
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 189
aatgatacgg cgaccaccga gatctacacc aacgtcttcg tcggcagcgt c 51
<210> 190
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 190
aatgatacgg cgaccaccga gatctacaca acacaggtcg tcggcagcgt c 51
<210> 191
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 191
aatgatacgg cgaccaccga gatctacact accaagctcg tcggcagcgt c 51
<210> 192
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 192
aatgatacgg cgaccaccga gatctacaca ggtggattcg tcggcagcgt c 51
<210> 193
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 193
aatgatacgg cgaccaccga gatctacacc atactgctcg tcggcagcgt c 51
<210> 194
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 194
aatgatacgg cgaccaccga gatctacacg atgcagttcg tcggcagcgt c 51
<210> 195
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 195
aatgatacgg cgaccaccga gatctacaca acagtcctcg tcggcagcgt c 51
<210> 196
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 196
aatgatacgg cgaccaccga gatctacact accttcgtcg tcggcagcgt c 51
<210> 197
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 197
aatgatacgg cgaccaccga gatctacacc atagacgtcg tcggcagcgt c 51
<210> 198
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 198
aatgatacgg cgaccaccga gatctacacg atggtcatcg tcggcagcgt c 51
<210> 199
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 199
aatgatacgg cgaccaccga gatctacaca actccactcg tcggcagcgt c 51
<210> 200
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 200
aatgatacgg cgaccaccga gatctacacg agtaggatcg tcggcagcgt c 51
<210> 201
<211> 51
<212> DNA
<213> Artificial sequence (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 (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 (Artificial Sequence)
<400> 208
aatgatacgg cgaccaccga gatctacaca acctggttcg tcggcagcgt c 51
<210> 209
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 209
aatgatacgg cgaccaccga gatctacact agctcactcg tcggcagcgt c 51
<210> 210
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 210
aatgatacgg cgaccaccga gatctacacg tagaacctcg tcggcagcgt c 51
<210> 211
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 211
aatgatacgg cgaccaccga gatctacact tcacacctcg tcggcagcgt c 51
<210> 212
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 212
aatgatacgg cgaccaccga gatctacacc acacgaatcg tcggcagcgt c 51
Claims (1)
1. A tag sequence set for second generation sequencing, comprising a nucleotide sequence as shown in SEQ ID No.1-SEQ ID No.142 and its reverse complement.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910779115.8A CN110468188B (en) | 2019-08-22 | 2019-08-22 | Tag sequence set for second generation sequencing and design method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910779115.8A CN110468188B (en) | 2019-08-22 | 2019-08-22 | Tag sequence set for second generation sequencing and design method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110468188A CN110468188A (en) | 2019-11-19 |
| CN110468188B true CN110468188B (en) | 2023-08-22 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201910779115.8A Active CN110468188B (en) | 2019-08-22 | 2019-08-22 | Tag sequence set for second generation sequencing and design method and application thereof |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444769B (en) * | 2020-03-28 | 2023-06-23 | 深圳人体密码基因科技有限公司 | Construction method and application of DNA tag sequence |
| CN113517026B (en) * | 2021-06-16 | 2022-08-19 | 苏州拉索生物芯片科技有限公司 | Method and system for generating label sequence applied to biological product, intelligent terminal and computer readable storage medium |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102115789A (en) * | 2010-12-15 | 2011-07-06 | 厦门大学 | Nucleic acid label for second-generation high-flux sequencing and design method thereof |
| WO2012037876A1 (en) * | 2010-09-21 | 2012-03-29 | 深圳华大基因科技有限公司 | Dna tag and application thereof |
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