CN107653299A - A kind of acquisition methods of the gene chip probes sequence based on high-flux sequence - Google Patents
A kind of acquisition methods of the gene chip probes sequence based on high-flux sequence Download PDFInfo
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- CN107653299A CN107653299A CN201610585938.3A CN201610585938A CN107653299A CN 107653299 A CN107653299 A CN 107653299A CN 201610585938 A CN201610585938 A CN 201610585938A CN 107653299 A CN107653299 A CN 107653299A
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Abstract
The present invention relates to biological technical field, and specifically a kind of acquisition methods of the gene chip probes sequence based on high-flux sequence, step include：(1) testing gene fragment is determined；(2) unknown species high-flux sequence to be measured；(3) high flux detection splicing；(4) blast comparisons are done using known species gene；(5) gene chip probes design.The present invention can significantly accelerate to obtain unknown species corresponding gene fragment to be checked, it compensate for the defects of Standard PCR can not be expanded effectively sometimes, simultaneously, background information can be increased by comparing the paralog gene found out or non-specific similar fragments, non-specific probe hybridization is effectively reduced, so as to reduce false positive.
Description
Technical field
The present invention relates to biological technical field, a kind of specifically gene chip probes sequence based on high-flux sequence
Acquisition methods.
Background technology
Genetic chip due to its have it is parallel, quickly, high flux detect the features such as, have extensively in molecular diagnostic techniques field
General utilization.Genetic chip is mainly by allowing the detected biological specimen gene probe exclusive with the inspection product on chip mutually to tie
Close, combined without probe corresponding with other inspection product, to judge whether the sample is that certain biotechnology for examining product detects product.It is this
The combination of inspection product and chip probe is carried out by way of nucleic acid hybridization, and the probe of this group-specific can be detected successfully
It is crucial.
The genetic chip most important is that the probe sequence for finding a group-specific can clearly distinguish measuring samples.
Under normal circumstances, the probe of this group-specific is made up of homologous gene, that is, the close sample detected all has this section of gene.
Due to the similitude of homologous gene, each probe has similitude, while the variant sites of gene again be present by these probe regions
Separate.In the genetic fragment and corresponding probe for initially selecting to be detected, it is necessary to come by gene pool (such as GenBank)
Find related background information.In practical operation, for specific one group of detection object, do not include institute many times in gene pool
Need to be detected the gene order of species.For these species to be checked without selected gene, generally we utilize general
The method that primer enters performing PCR amplification finds out homologous gene corresponding to the species.
It is but this to be faced with two large problems with the method for standard PCR amplification in practice.First, as chip
Probe will have enough difference, and it may also make a variation in the position of primer.Thus, custom primer can not be expanded effectively
These homologous genes, so as to which the sequence and probe of the species can not be obtained.By other means obtain the method for the gene, such as
Genomic walking or RACE (cDNA ends rapid amplifying) technology, often get half the result with twice the effort.Secondly, as detection range expands, institute
The genetic test scope of selection is also increasingly wider, and much gene is in addition to ortholog, and also there is paralog
Gene (non-specific gene).The not only possible amplification for influenceing ortholog of the sequence of these paralog genes, and
Final hybridization may be made the result of false positive occur.
High throughput sequencing technologies flourish at present, and the growth of blowout occurs in gene sequencing.Utilize high-flux sequence
Technology, ortholog can effectively and be quickly found, while obtaining paralog gene or non-specific phase
Like fragment, so as to exclude the interference of these paralog genes when probe designs.
The content of the invention
The purpose of the present invention is the problem of presence for background technology, there is provided one kind is obtained not based on high throughput sequencing technologies
Inspection product coherent detection gene order is known, so as to carry out the screening of probe and exclude the interference of other non-specific genes.
To achieve the above object, the technical solution adopted by the present invention is：A kind of genetic chip based on high-flux sequence is visited
The acquisition methods of pin sequence, step include：
(1) testing gene fragment is determined：According to existing data, evaluate for the gene core needed for particular detection project
Piece target gene fragment；
(2) unknown species high-flux sequence to be measured：To needing to detect but not having the species or sample progress height of the gene
Flux is sequenced, and different high-flux sequence schemes can be used according to the purpose of detection；
(3) high flux detection splicing：High-flux sequence final finishing, the software using correlation are spliced, and generate contig
File, analyzed for follow-up comparison；
(4) blast comparisons are done using known species gene：With known this section of gene of species, generated in splicing
Contig files compare, and directly fish out the unknown gene；
(5) gene chip probes design：The gene order design length according to the different plant species collected is 18-40bp
Probe.
Further, in step (2), the detection for specific coding gene takes mesh using the scheme of transcript profile sequencing
Particular organization's sample of species is marked, extracts RNA, after reverse transcription is cDNA, high-flux sequence is carried out and builds storehouse, be then sequenced；
Further, in step (2), for gene flanking sequence and the simplified gene order-checking of non-coding sequence detection
Scheme, i.e., the DNA of target species or sample is directly extracted, builds storehouse, then high-flux sequence.
Further, in step (3), transcript profile sequencing is spliced using Trinity softwares.
Further, in step (3), simplify gene order-checking and spliced using SOAPdenovo softwares.The present invention's
Advantageous effects are：The present invention can significantly accelerate to obtain unknown species corresponding gene fragment to be checked, compensate for routine
The defects of PCR can not be expanded effectively sometimes, meanwhile, comparing the paralog gene found out or non-specific similar fragments can increase
Background information, non-specific probe hybridization is effectively reduced, so as to reduce false positive.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 is the flow chart of the inventive method.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Embodiment 1
As shown in figure 1, a kind of acquisition methods of the gene chip probes sequence based on high-flux sequence, step include：
(1) according to existing data, evaluate for the genetic chip target gene fragment needed for particular detection project.
(2) to needing to detect but not having the species or sample progress high-flux sequence of the gene；According to the purpose of detection
Two kinds of different high-flux sequence schemes can be used：
1. the scheme that the detection for specific coding gene is sequenced using transcript profile, that is, take the particular organization of target species
Sample, RNA is extracted, after reverse transcription is cDNA, carries out high-flux sequence and build storehouse, be then sequenced；
2. the scheme for simplifying gene order-checking can be used for gene flanking sequence and non-coding sequence detection, i.e., directly carry
The DNA of target species or sample is taken, builds storehouse, then high-flux sequence.
(3) high-flux sequence final finishing, the software using correlation are spliced, and contig files are generated, for follow-up
Compare analysis.
Different joining methods is used for different sequencing schemes：Trinity softwares can be used to carry out for transcript profile sequencing
Splicing；Simplify gene order-checking using SOAPdenovo softwares to be spliced.
(4) this section of gene of species known to using, compared in the contig files of splicing generation, directly fish out the unknown base
Cause, used software are Blast+.Concrete operations are：First by contig file generateds blast needed for formatted data base；
Then this section of gene of species is source file known to, is found out similar to the genetic fragment in the unknown species database established
Sequence, arrange parameter-evalue is 1e-5, and-num_alignments is 250, makes to occur in output file a plurality of with its phase
As sequence；In output file, evalue value highests, and gene is then defined as direct line together for the sequence of clear and definite encoding gene
The target gene in source, and the other sequences found out are paralog gene (non-specific fragment).
(5) probe is designed according to the gene order for the different plant species collected.Concrete operations are：Collected all
The genetic fragment and non-specific fragment for needing detection species carry out Clustal comparisons together, using MEGA or Clustal
The softwares such as X are completed；After comparison, the special probe of 2-3 sections, probe length 18- are found out from the target gene of each species
40bp；These probes calls are that the species are peculiar, have difference with other species sequences and non-specific fragment.
Embodiment 2
Amphibian animal Sex Determination and development detection genetic chip use.In amphibian animal, Dmrt1 genes and Sex Determination and
Development is related, but also not comprising many amphibian genes in gene pool.Therefore, Rana boulengeri and Sichuan to that need to detect
The rapid frog carries out transcript profile sequencing, after Trinity splicings, by known Xenopus laevis and Rana nigromaculata Dmrt1 genes, is found out through blast
The Dmrt1 genes of the two species and non-specific sequence.Finally by collected need detect species the genetic fragment and
Non-specific fragment carries out Clustal comparisons together, finds out the probe that 2-3 sections are special in the target gene of each species.
Embodiment 3
Quarantine fruit fly DNA bar code detects genetic chip application.DNA bar code, i.e. COI gene orders are widely used as
The identification of species, but because the gene is chondriogen, it has similar to the gene non-specific in Matrix attachment region
Gene (pseudogene, numt).These non-specific genetic fragments disturb DNA bar code application, raise false positive.Therefore, choosing
5 kinds of trypetids (tool bar trypetid, citrus fruit fly, pumpkin fruit fly, different face trypetid, melonfly) for being not easy to recognize have been taken to carry out simplifying gene
Group sequencing.After splicing, with known above-mentioned species COI genes, the COI genes of these species and non-specific sequence are found out through blast
Arrange (pseudogene).Finally the genetic fragment for needing to detect species collected and non-specific fragment are carried out together
Clustal is compared, and finds out the probe that 2-3 sections are special in the target gene of each species.Avoid it is identical with non-specific sequence,
So as to reduce false positive.
Those skilled in the art of the present technique are appreciated that unless otherwise defined, all terms used herein (including technology art
Language and scientific terminology) with the general understanding identical meaning with the those of ordinary skill in art of the present invention.Should also
Understand, those terms defined in such as general dictionary, which should be understood that, to be had and the meaning in the context of prior art
The consistent meaning of justice, and unless defined as here, will not be with idealizing or the implication of overly formal be explained.
It should be noted last that：Above example is only to illustrative and not limiting technical scheme, although ginseng
The present invention is described in detail according to above-described embodiment, it will be apparent to an ordinarily skilled person in the art that：Still can be to this
Invention is modified or equivalent substitution, and any modification or partial replacement without departing from the spirit and scope of the present invention, its is equal
It should cover among scope of the presently claimed invention.
Claims (5)
1. a kind of acquisition methods of the gene chip probes sequence based on high-flux sequence, it is characterised in that step includes：
(1) testing gene fragment is determined：According to existing data, evaluate for the genetic chip mesh needed for particular detection project
Mark genetic fragment；
(2) unknown species high-flux sequence to be measured：To needing to detect but not having the species or sample progress high flux of the gene
Sequencing, different high-flux sequence schemes can be used according to the purpose of detection；
(3) high flux detection splicing：High-flux sequence final finishing, the software using correlation are spliced, and generate contig files,
Analyzed for follow-up comparison；
(4) blast comparisons are done using known species gene：It is literary in the contig of splicing generation with known this section of gene of species
Part compares, and directly fishes out the unknown gene；
(5) gene chip probes design：Visited according to the gene order design length for the different plant species collected for 18-40bp
Pin.
2. the acquisition methods of the gene chip probes sequence based on high-flux sequence according to claim 1, it is characterised in that
In step (2), the detection for specific coding gene uses the scheme of transcript profile sequencing, that is, takes the particular organization of target species
Sample, RNA is extracted, after reverse transcription is cDNA, carries out high-flux sequence and build storehouse, be then sequenced.
3. the acquisition methods of the gene chip probes sequence based on high-flux sequence according to claim 1, it is characterised in that
In step (2), for gene flanking sequence and the non-coding sequence detection scheme for simplifying gene order-checking, i.e., mesh is directly extracted
The DNA of species or sample is marked, builds storehouse, then high-flux sequence.
4. the acquisition methods of the gene chip probes sequence according to claim 1 or claim 2 based on high-flux sequence, its feature exist
In in step (3), transcript profile sequencing is spliced using Trinity softwares.
5. the acquisition methods of the gene chip probes sequence based on high-flux sequence, its feature exist according to claim 1 or 3
In in step (3), simplified gene order-checking is spliced using SOAPdenovo softwares.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021023142A1 (en) * | 2019-08-02 | 2021-02-11 | 华为技术有限公司 | Gene alignment technique |
| CN112513292A (en) * | 2018-08-27 | 2021-03-16 | 深圳华大生命科学研究院 | Method and device for detecting homologous sequence based on high-throughput sequencing |
| CN117012274A (en) * | 2023-10-07 | 2023-11-07 | 北京智因东方转化医学研究中心有限公司 | Device for identifying gene deletion based on high-throughput sequencing |
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| JP2008118921A (en) * | 2006-11-13 | 2008-05-29 | Canon Inc | Method for identification of biological species |
| CN105274092A (en) * | 2015-11-30 | 2016-01-27 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Batch acquiring method for specific isothermal oligonucleotide probes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112513292A (en) * | 2018-08-27 | 2021-03-16 | 深圳华大生命科学研究院 | Method and device for detecting homologous sequence based on high-throughput sequencing |
| CN112513292B (en) * | 2018-08-27 | 2023-12-26 | 深圳华大生命科学研究院 | Method and device for detecting homologous sequences based on high-throughput sequencing |
| WO2021023142A1 (en) * | 2019-08-02 | 2021-02-11 | 华为技术有限公司 | Gene alignment technique |
| CN117012274A (en) * | 2023-10-07 | 2023-11-07 | 北京智因东方转化医学研究中心有限公司 | Device for identifying gene deletion based on high-throughput sequencing |
| CN117012274B (en) * | 2023-10-07 | 2024-01-16 | 北京智因东方转化医学研究中心有限公司 | Device for identifying gene deletion based on high-throughput sequencing |
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