CN106755542A - The bioinformatic analysis method of peripheral blood dissociative DNA deep sequencing result - Google Patents
The bioinformatic analysis method of peripheral blood dissociative DNA deep sequencing result Download PDFInfo
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- CN106755542A CN106755542A CN201710129339.5A CN201710129339A CN106755542A CN 106755542 A CN106755542 A CN 106755542A CN 201710129339 A CN201710129339 A CN 201710129339A CN 106755542 A CN106755542 A CN 106755542A
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Abstract
The invention provides a kind of bioinformatic analysis method of peripheral blood dissociative DNA deep sequencing result, comprise the following steps：1) primer label is designed；2) primer label is connected to each DNA molecular；3) performing PCR amplification, high-flux sequence are entered；4) peripheral blood dissociative DNA deep sequencing result is obtained by bioinformatic analysis.The present invention is by giving each tagged mode in initial sample template dna molecule both sides, molecular label is sticked to each DNA molecular in template, after PCR and sequencing, with reference to analysis of biological information method, discriminating classification, the systematic error that PCR amplifications and sequencing stage introduce in removal or reduction NGS sequencings are carried out to DNA molecular original in sample using molecular label.
Description
Technical field
The present invention relates to high-flux sequence field, and in particular to a kind of biology of peripheral blood dissociative DNA deep sequencing result
Bioinformatics analysis method.
Background technology
CtDNA contents are considerably less, by taking 10ml peripheral bloods as an example, can therefrom extract cfDNA contents about 30ng, account for total
Ratios of 1%, the ctDNA of DNA in all cfDNA is considerably less, is calculated by 1%, only about 300pg, that is, about 45
The amount of DNA of cell cracking, under actual conditions, if doing early screening, the content of ctDNA may be in the lower peripheral blood to 10ml
Only 1~2 cell, so very high to the sensitive requirements of detection method.
Method currently used for target gene detection in liquid biopsy is broadly divided into four classes：A generation is sequenced (Sanger), and two
For high-flux sequence (NGS), digital drop PCR (ddPCR), and ARMS (also known as ApoE gene).For tumour base
For the examination of cause, NGS detects that the cost performance in similar far exceeds other several methods.
NGS methods can be related to PCR amplifications and be sequenced, and some system mistakes can all occur in the two processes, such as
, 0.05%, problem is simultaneously little in common great amount of samples template detection for the sequencing error rate of illumina Hiseq platforms, but
It is, in tumour early screening, because template class itself is exactly these sequencings within the scope of a ten thousandth to one thousandth
Mistake is offline in an order of magnitude with our detection, very serious interference will be caused to result, secondly as PCR draws
The mistake for entering also cannot be recognized effectively.
Chinese patent application (application number CN201510225771.5) discloses a kind of fetal cell-free DNA in maternal plasma
The quantitative approach of ratio, the method is extracted the DNA comprising design SNP in blood plasma using the primer of design, is by one
Upper machine after column processing, will be sequenced product and compares to after human genomic sequence the base for determining each SNP site.By analysis
The ratio of each base quantifies fetus dissociative DNA concentration in SNP.The present invention can also correct the error of introducing by multiple SNP,
Multiple SNP are calculated simultaneously with statistical method, the fetus dissociative DNA concentration accuracy that raising is calculated.
However, purely improving the sequencing degree of accuracy by improving single analysis of biological information method, pertain only to detect number
Processed according to the later stage, the systematic error that can not be introduced when source eliminates or effectively reduce PCR amplifications and is sequenced.
So be badly in need of developing a kind of new method at present, for differentiating real gene mutation and these system mistakes.
The content of the invention
To solve the above problems, the invention provides a kind of bioinformatics of peripheral blood dissociative DNA deep sequencing result
Analysis method.
In a first aspect, the invention provides a kind of bioinformatic analysis side of peripheral blood dissociative DNA deep sequencing result
Method, comprises the following steps：
1) primer label is designed；
2) primer label is connected to each DNA molecular；
3) performing PCR amplification, high-flux sequence are entered；
4) peripheral blood dissociative DNA deep sequencing result is obtained by bioinformatic analysis.
Deep sequencing method of the present invention, can be those skilled in the art and routinely understand, such as with high-flux sequence skill
The concepts such as art, NGS are interchangeable as a rule.
The sequencing equipment that deep sequencing method of the present invention is used includes but is not limited to Roche/454, Illumina survey
Sequence instrument (NextSeq series, Hiseq series, MiSeq series, XTen, and instrument family is subsequently sequenced), BGI (Hua Da company,
BGI500 series and follow-up sequenator) sequenator, LifeTech sequencings instrument (Ion, Proton and instrument is subsequently sequenced
Series), PacBio sequencings instrument (RSII, Sequel and instrument is subsequently sequenced) or the sequencing instrument based on Nanopore
(Genia, Nanopore and similar third generation sequenator).
Second aspect, cancer is expanded the invention provides a kind of primer label combination deep sequencing method from peripheral blood dissociative DNA
The label method for designing of disease gene, comprises the following steps：
1) primer label is designed；
2) primer label is connected to each DNA molecular in template；
3) PCR primer or PCR primer group are designed for Target cancers gene；
4) enter performing PCR amplification and be sequenced, filter out qualified primer label.
What the present invention was provided expands the basic principle of the molecular label design of cancer gene from peripheral blood dissociative DNA：It is logical
Cross and give each tagged mode in initial sample template both sides, molecular label is sticked to each DNA molecular in template, in warp
Cross after PCR and sequencing, DNA molecular original in sample can all be differentiated by classification by molecular label.Due to expanding by PCR
Increase later molecule and all carry same molecular label with primary template, so in theory, from same molecule mark
Certain the class DNA molecular signed should carry identical sequence, if the mutation that the product in the middle of the later stage occurs during PCR, just
These mutant nucleotide sequences can be removed by label, it is to avoid they disturb last sequencing result.Likewise, sequencing mistake can also
Differentiated by this method.
The third aspect, the invention provides a kind of for deep sequencing method from peripheral blood dissociative DNA amplification cancer gene
Primer label.
Fourth aspect, the invention provides a kind of primer sets, primer that such as second aspect is provided provided such as first aspect
Label method for designing or the primer label provided such as the third aspect are expanding answering in cancer gene from peripheral blood dissociative DNA
With or application in high-flux sequence.
Brief description of the drawings
Fig. 1 is a kind of bioinformatic analysis of peripheral blood dissociative DNA deep sequencing result provided in an embodiment of the present invention
Method flow schematic diagram.
Specific embodiment
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
In the embodiment of the present invention unless otherwise noted, agents useful for same and consumptive material are commercial goods.
If being illustrated in the embodiment of the present invention, sequencing library builds and is measured with reference to Roche, the high pass of illumina or ABI
Sequence library construction specification.
In the first embodiment of the invention, with reference to Fig. 1, the invention provides a kind of peripheral blood dissociative DNA deep sequencing knot
The bioinformatic analysis method of fruit, comprises the following steps：
1) primer label is designed；
2) primer label is connected to each DNA molecular in template；
3) PCR primer or PCR primer group are designed for Target cancers gene；
4) performing PCR amplification, high-flux sequence are entered；
5) peripheral blood dissociative DNA deep sequencing result is obtained by bioinformatic analysis.
The sequencing equipment that deep sequencing method of the present invention is used includes but is not limited to Roche/454, Illumina survey
Sequence instrument (NextSeq series, Hiseq series, MiSeq series, XTen, and instrument family is subsequently sequenced), BGI (Hua Da company,
BGI500 series and follow-up sequenator) sequenator, LifeTech sequencings instrument (Ion, Proton and instrument is subsequently sequenced
Series), PacBio sequencings instrument (RSII, Sequel and instrument is subsequently sequenced) or the sequencing instrument based on Nanopore
(Genia, Nanopore and similar third generation sequenator).
In second embodiment of the invention, swum from peripheral blood the invention provides a kind of primer label combination deep sequencing method
From the label method for designing of DNA cloning cancer gene, comprise the following steps：
1) primer label is designed；
2) primer label is connected to each DNA molecular in template；
3) PCR primer or PCR primer group are designed for Target cancers gene；
4) enter performing PCR amplification and be sequenced, filter out qualified primer label.
What the present invention was provided expands the basic principle of the molecular label design of cancer gene from peripheral blood dissociative DNA：It is logical
Cross and give each tagged mode in initial sample template both sides, molecular label is sticked to each DNA molecular in template, in warp
Cross after PCR and sequencing, DNA molecular original in sample can all be differentiated by classification by molecular label.Due to expanding by PCR
Increase later molecule and all carry same molecular label with primary template, so in theory, from same molecule mark
Certain the class DNA molecular signed should carry identical sequence, if the mutation that the product in the middle of the later stage occurs during PCR, just
These mutant nucleotide sequences can be removed by label, it is to avoid they disturb last sequencing result.Likewise, sequencing mistake can also
Differentiated by this method.
In third embodiment of the invention, expand from peripheral blood dissociative DNA for deep sequencing method the invention provides one kind
Increase the primer label of cancer gene.
In fourth embodiment of the invention, the invention provides a kind of such as the primer sets of first aspect offer, such as second party
The primer label method for designing that face provides or the primer label that such as third aspect is provided are expanding cancer from peripheral blood dissociative DNA
Application in disease gene or the application in high-flux sequence.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (4)
1. a kind of bioinformatic analysis method of peripheral blood dissociative DNA deep sequencing result, comprises the following steps：
1) primer label is designed；
2) primer label is connected to each DNA molecular；
3) performing PCR amplification, high-flux sequence are entered；
4) peripheral blood dissociative DNA deep sequencing result is obtained by bioinformatic analysis.
2. a kind of primer label combination deep sequencing method expands the label method for designing of cancer gene from peripheral blood dissociative DNA, its
It is characterised by, comprises the following steps：
1) primer label is designed；
2) primer label is connected to each DNA molecular in template；
3) PCR primer or PCR primer group are designed for Target cancers gene；
4) enter performing PCR amplification and be sequenced, filter out qualified primer label.
3. it is a kind of for deep sequencing method from peripheral blood dissociative DNA expand cancer gene primer label.
4. a kind of bioinformatic analysis method of peripheral blood dissociative DNA deep sequencing result as claimed in claim 1, such as weigh
Profit require primer label method for designing described in 2 or primer label as claimed in claim 3 in high-flux sequence should
With.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710454A (en) * | 2013-12-31 | 2014-04-09 | 南方科技大学 | Method for carrying out high-throughput sequencing on TCR (T cell receptor) or BCR (B cell receptor) and method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing tag sequences |
| CN105331606A (en) * | 2014-08-12 | 2016-02-17 | 焦少灼 | Nucleic acid molecule quantification method applied to high-throughput sequencing |
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2017
- 2017-03-06 CN CN201710129339.5A patent/CN106755542A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710454A (en) * | 2013-12-31 | 2014-04-09 | 南方科技大学 | Method for carrying out high-throughput sequencing on TCR (T cell receptor) or BCR (B cell receptor) and method for correcting multiplex PCR (polymerase chain reaction) primer deviation by utilizing tag sequences |
| CN105331606A (en) * | 2014-08-12 | 2016-02-17 | 焦少灼 | Nucleic acid molecule quantification method applied to high-throughput sequencing |
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Effective date of registration: 20190902 Address after: 518101 Tingwei Industrial Park Building 301, No. 6, Liufang Road, 67 District, Xin'an Street, Baoan District, Shenzhen City, Guangdong Province Applicant after: Shenzhen Yinhe Biotechnology Co., Ltd. Address before: 518052 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A Applicant before: From Shenzhen Biological Technology Co. Ltd. |
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