CN110468188A - For the sequence label collection and its design method of the sequencing of two generations and application - Google Patents
For the sequence label collection and its design method of the sequencing of two generations and application Download PDFInfo
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- CN110468188A CN110468188A CN201910779115.8A CN201910779115A CN110468188A CN 110468188 A CN110468188 A CN 110468188A CN 201910779115 A CN201910779115 A CN 201910779115A CN 110468188 A CN110468188 A CN 110468188A
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- sequencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The present invention relates to a kind of sequence label collection for the sequencing of two generations and its design method and applications, belong to technical field of molecular biology.It is 6-10 that the sequence label, which concentrates the length of each sequence label, and the G/C content in each sequence label is 40-60%, and continuous base number is up to 1-3 in each sequence label, and the first two base of each sequence label is not G simultaneously.It is sequenced using above-mentioned sequence label collection for two generations, pass through the screening and combination to sequence label, make finally to obtain sequence label collection quantity it is higher, it can be achieved that large sample size detection requirement, and can require while can correctly split out the sequencing data of high quality meet sequencing.
Description
Technical field
The present invention relates to molecular biology fields, more particularly to a kind of sequence label collection for the sequencing of two generations and its set
Count methods and applications.
Background technique
The sequencing of two generations (also known as next-generation sequencing, NGS) technology has been increasingly becoming in all various aspects of discovery and Study on Transformation
A technique for it being widely adopted, the sequencing of two generations can simultaneously be sequenced multiple samples simultaneously due to high-throughput characteristic,
And the method for distinguishing sample is exactly that sequence label (index) is used to distinguish.
The sequence that sequence label mostly uses greatly microarray dataset (such as Illumina) to provide at present, but what microarray dataset was given
Sequence label Limited Number, then carry out just needing further amounts of label sequence when extensive sample sequencing or both-end sequencing
Column.
Current individually designed sequence label lack a kind of method of the tag design sequence of system and based on stringent screening and
The sequence label of generation, the sequence label of oneself synthesis are possible to not meet sequencing requirement and lead to sequencing failure or sequencing
Data are difficult to split.
Summary of the invention
Based on this, it is necessary in view of the above-mentioned problems, providing a kind of sequence label collection for the sequencing of two generations, such label sequence
Column can meet the sequencing data that requirement is sequenced while can correctly split out high quality.
A kind of sequence label collection for the sequencing of two generations, it is 6- that the sequence label, which concentrates the length of each sequence label,
10, the G/C content in each sequence label is 40-60%, and continuous base number is up to 1-3, each label in each sequence label
The first two base of sequence is not G simultaneously.
The sequence label that above-mentioned sequence label is concentrated, can be used for library construction, is sequenced followed by microarray dataset.It can manage
Solution, the sequence label of design is used on reverse primer such as, needs to carry out reverse complemental to sequence label.
The length of the sequence label is 8 in one of the embodiments, and continuous base number is maximum in each sequence label
It is 2.Be 8 by the control of the length of sequence label, since segment is longer, serious forgiveness is bigger, but long segment also imply that it is longer
Time and the consumption of more reagents is sequenced, considers from practice, it is a preferable length that maximum length, which is controlled as 8,；Same time control
It makes its continuous base number and is up to 2, can be reduced while guaranteeing base contents equilibrium to greatest extent because sequencing generates
Base misread.
The G/C content of the sequence label is 50% in one of the embodiments,.G/C content is the bird in 4 kinds of bases
Ratio shared by purine (G) and cytimidine (C).
Each sequence label that the sequence label is concentrated in one of the embodiments, is compared two-by-two, for
The base of same position, identical to be denoted as 0 point, different is denoted as 1 point, calculates total score, and the total score of each sequence label >=
4.The above-mentioned purpose for determining sequence label with the limit of integration is to increase the Hamming distance (Hamming between two labels
Distance), more preferable two sequences must can be distinguished.For example, when two sequences are respectively as follows: TGGTTACC, CAACCAGA, only
6th base is the same, is all " A ", so label total score is 7, and two sequences for another example: CGATCACA, CAATCAGA, label are total
Be divided into 2, it is seen that the difference of previous group be greater than later group, can better distinguishing sequence, the mistake of identification is generated due to mispairing
It substantially reduces.
The total score of each sequence label is 4 in one of the embodiments,.Have the advantages that have a little as follows: 1, it is maximum
The otherness of the guarantee sequence label of limit；2, the sequence label for being sufficiently used for production can be obtained.
The sequence label is selected from as shown in SEQ ID No.1-SEQ ID No.142 in one of the embodiments,
Nucleotide sequence.Above-mentioned sequence label can meet the sequencing data that requirement is sequenced while can correctly split out high quality.
Shown in table specific as follows:
1. sequence label of table
Base contents ratio of the sequencing label in same position in one of the embodiments, are as follows: A=20-30%, B
=20-30%, C=20-30%, D=20-30%.By the base contents control of same position in above range, have following excellent
Point: it both can guarantee that base balanced, the light of the excessively high generation of some signals of same position avoided to spread out；It can avoid some letters of same position again
Number too low and skip.
The invention also discloses the above-mentioned sequence label collection for the sequencing of two generations pathogenic microorganism DNA and/or RNA
Application in PCR product sequencing.
The invention also discloses the design methods of the above-mentioned sequence label collection for the sequencing of two generations, comprising the following steps:
1) sequence of all base compositions of preset label length is listed；
2) sequence for wherein meeting predetermined G/C content is selected；
3) sequence that continuous base number in above-mentioned sequence is up to 2 is screened；
4) going out the first two base in the above-mentioned sequence of screening is simultaneously the sequence of G；
5) above-mentioned sequence is compared two-by-two, for the base of same position, identical to be denoted as 0 point, different is denoted as 1
Point, total score is calculated, the sequence of total score >=4 is selected；
6) according to required sequence label quantity, sequence is selected in above-mentioned sequence, makes gained sequencing label in same position
Base contents ratio are as follows: A=20-30%, B=20-30%, C=20-30%, D=20-30%.
Compared with prior art, the invention has the following advantages:
A kind of sequence label collection for the sequencing of two generations of the invention is made most by the screening and combination to sequence label
Eventually obtain sequence label collection quantity it is higher, it can be achieved that large sample size detection requirement, and can meet sequencing require simultaneously energy
Enough sequencing datas for correctly splitting out high quality.
Specific embodiment
To facilitate the understanding of the present invention, below with reference to embodiment to invention is more fully described.But this hair
It is bright to realize in many different forms, however it is not limited to embodiment described herein.On the contrary, providing these embodiments
Purpose be to make the disclosure of the present invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
Embodiment 1
The sequencing of two generations is carried out using sequence label collection of the invention, steps are as follows:
1, synthetic linker primer.
According to following table synthetic linker primer, and it is diluted to 10 μM.
2. primer sequence table of table
Note: underscore part indicates sequence label in upper table sequence, and wherein SEQ ID No.146-148 is using reversed
Complementary series.
2, sample extraction.
According to Qiagen QIAamp Viral RNA Mini Kit, (article No.: 52906) method extracts different sample types
RNA, with 3.0 fluorescent quantitation instrument accurate quantitative analysis RNA concentration of specimens of Qubit.As a result as shown in the table.
Each sample extraction of table 3. and selection index
3, library construction and upper machine are sequenced.
Library kit (NR603) is built with VAHTS RNA to carry out after RNA library construction on Illumina NEXTSEQ550
Machine testing.The Quality Control of machine data result is as shown in the table on gained.
Machine data result Quality Control result table on table 4.
| 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 |
It can be seen from the results above that Q30 > 93% of data, Adaptor ratio less than 5%, show in extremely low initial amount
In the case where still meet Jian Ku and upper confidential ask.
Embodiment 2 (double index)
The sequencing of two generations is carried out using sequence label collection of the invention, substantially the same manner as Example 1, difference is, is used
Sequence label it is as shown in the table.
5. primer sequence table of table
Note: underscore part indicates sequence label in upper table sequence, and wherein SEQ ID No.149-180 is using reversed
Complementary series.
Sequencing approach:
1, the adapter-primer in table 5 is synthesized, and is diluted to 10 μM of
2, sample extraction.
According to Beijing Tiangeng biochemistry Co., Ltd trace sample genome DNA extracting reagent kit (DP316) method, brain is extracted
The DNA of spinal fluid, with 3.0 fluorescent quantitation instrument accurate quantitative analysis RNA concentration of specimens of Qubit.As a result as shown in the table.
Each sample extraction of table 6. and selection index
3, library construction and upper machine are sequenced.
Library kit IndexuePrep DNA Library Prep Kit V2 for Illumina is built with VAHTS
(TD503-01) machine is sequenced on Illumina NEXTSEQ550 after progress DNA library building.Machine data result Quality Control on gained
As shown in the table.
7. Quality Control result of table
| 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 |
8. data of table split size
It can be seen from the results above that Q30 > 95% of data, Adaptor ratio are less than the data that 2%, i5 and i7 are split out
Equilibrium shows that designed joint sequence can satisfy the requirement for building library on a large scale.
In conclusion sequence label collection of the invention can overcome existing number of labels few, it is difficult to realize that large sample size is same
When detect, simultaneous failure tolerance rate is low, be easy to cause data split mistake, lead to the problem of false positive or false negative.And part
That there are continuous sequences is too long for sequence label, and sequencing procedure is difficult to interpretation and GC base balance is poor, and it is poor to easily lead to the quality of data
The problems such as.Simultaneously by designing suitable Hamming distance, the error rate that machine reads base is reduced.Both there is sequence label collection
Quantity it is big, it can be achieved that large sample size detection requirement, and can be required meet sequencing while can correctly split out high quality
Sequencing data the advantages of.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Guangzhou Wei Yuan Gene Tech. Company Limited
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ctactgca 8
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atcctctg 8
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tcatgtcg 8
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ctagagag 8
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gcaactgt 8
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atcgatcg 8
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tcacactc 8
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ctagtctc 8
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gcagacat 8
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tcactgag 8
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cttccaag 8
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gcagtgta 8
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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 (9)
1. a kind of sequence label collection for the sequencing of two generations, which is characterized in that the sequence label concentrates each sequence label
Length is 6-10, and the G/C content in each sequence label is 40-60%, and continuous base number is up to 1-3 in each sequence label,
The first two base of each sequence label is not G simultaneously.
2. the sequence label collection according to claim 1 for being used for the sequencing of two generations, which is characterized in that the length of the sequence label
Degree is 8, and continuous base number is up to 2 in each sequence label.
3. the sequence label collection according to claim 1 for being used for the sequencing of two generations, which is characterized in that the GC of the sequence label
Content is 50%.
4. the sequence label collection according to claim 1 for being used for the sequencing of two generations, which is characterized in that by the sequence label collection
In each sequence label compared two-by-two, for the base of same position, identical to be denoted as 0 point, different is denoted as 1 point,
Calculate total score, total score >=4 of each sequence label.
5. the sequence label collection according to claim 4 for being used for the sequencing of two generations, which is characterized in that each sequence label
Total score be 4.
6. the sequence label collection according to claim 1 for being used for the sequencing of two generations, which is characterized in that the sequence label is selected from
Nucleotide sequence and its reverse complementary sequence as shown in SEQ ID No.1-SEQ ID No.142.
7. the sequence label collection according to claim 1 for being used for the sequencing of two generations, which is characterized in that the sequencing label is in phase
With the base contents ratio of position are as follows: A=20-30%, B=20-30%, C=20-30%, D=20-30%.
8. claim 1-7 is described in any item be used for the sequence label collection of two generations sequencing in the DNA of pathogenic microorganism and/or
Application in the sequencing of RNA PCR product.
9. the design method of the described in any item sequence label collection for being used for the sequencing of two generations of claim 1-7, which is characterized in that packet
Include following steps:
1) sequence of all base compositions of preset label length is listed；
2) sequence for wherein meeting predetermined G/C content is selected；
3) sequence that continuous base number in above-mentioned sequence is up to 2 is screened；
4) going out the first two base in the above-mentioned sequence of screening is simultaneously the sequence of G；
5) above-mentioned sequence is compared two-by-two, for the base of same position, identical to be denoted as 0 point, different is denoted as 1 point,
Total score is calculated, the sequence of total score >=4 is selected；
6) according to required sequence label quantity, sequence is selected in above-mentioned sequence, makes gained sequencing label in the alkali of same position
Base content ratio are as follows: A=20-30%, B=20-30%, C=20-30%, D=20-30%.
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 |
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