CN106778060B - A method of it is completed figure using prokaryotic gene group high quality sketch - Google Patents
A method of it is completed figure using prokaryotic gene group high quality sketch Download PDFInfo
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Abstract
It is completed the method for figure the invention discloses a kind of using prokaryotic gene group high quality sketch, the process employs the principles of graph theory, its node and side are redefined to the sketch of high-flux sequence, corresponding network is established on this basis, corresponding sequence read in the gap of hole is excavated in prokaryotic gene group high quality sketch between contig from the network again, finally collects the sequence read in each hole and carries out local assembling and obtain corresponding genome completion figure；It is compared with the traditional method, the present invention does not need design primer and any additional experiment only needs to operate on computers using this method, avoids a large amount of experimentally cost while also greatly improving the efficiency；In addition, the prokaryotic gene group completion figure that present invention comparison conventional method obtains is compared to just the same.
Description
Technical field
The present invention relates to Bioinformatics technical field more particularly to a kind of utilization prokaryotic gene group high quality grass
Figure completes the method for figure.
Background technique
Currently, present invention employs the methods that bioinformatic data excavates to substitute traditional PCR and Sanger sequencing in fact
Proved recipe method.It is compared with the traditional method, the present invention does not need additional experimental cost while can save many experiments time.It obtains
The result that prokaryotic gene group completion figure and conventional method obtain is just the same.It is new that technology platform is widely used in prokaryotes
The genome de novo sequencing field of species.For the genome of prokaryotes new species, each high-flux sequence needs to carry out two
It is secondary: 1, interrupt to genome the segment of separation 600bp or so, carry out the high pass that 2x300bp sequencing depth is about 30x and measure
Sequence；2, the segment for interrupt to genome separation 8kb or so be cyclized-interrupting-and separates the linker's containing connection after being enriched with
The segment of 600bp or so carries out the high-flux sequence that the sequencing depth of 2x300bp is about 20x；In conjunction with sequencing data twice
Assemble the genome high quality sketch of the available prokaryotes.High quality sketch can provide a large amount of related protokaryon
The gene information of biology, can play an important role in the quick diagnosis such as diseases monitoring field.
But in the field that research associated biomolecule is evolved, the analysis of genome collinearity is particularly significant；Prokaryotes exist
In evolution, tends to situations such as large stretch of gene delection, rearrangement and transfer occur or even these evolution situations go out than individual gene
The frequencies such as now transfer, mutation, recombination are equally matched.Therefore, the biological information provided for high quality draft genome can only be right
Individual gene or small range collinearity region are studied, and the research of the comprehensive collinearity of genome relies only on base at present
Because of a group completion figure.In addition, the acquisition of genome completion figure will be significantly larger than high quality gene on experimental cost and time cost
Group sketch.
On NCBI Genbank, have at present about 3500 or so prokaryotic gene group completion figure and 300,000 left sides
Right prokaryotic gene group high quality sketch (in July, 2016), this quantitative comparison are enough to illustrate that genome completion figure is difficult to obtain
?.It is exactly that cost is excessively high using the greatest drawback that conventional method obtains prokaryotic gene group completion figure, and with
Contig quantity increases and corresponding cost will steeply rise to geometric progression.
For the high-flux sequence of prokaryotic gene group, tend to each in covering whole gene group while genome
The average sequencing depth of base can reach about 30x.However, to initial data carry out from the beginning assemble after, assembling result often by
Many contig contig compositions, different contig contig constitute scaffold again, the result is that a large amount of hole gap are left,
It needs to be remedied with PCR and Sanger sequencing experiment, the final genome that obtains completes figure.In fact, from the beginning assembling in result
Face is other than scaffold and the longer contig of sequence, and there are also the shorter contig of a large amount of sequence, and this kind of sequence is shorter
Contig also belong to the genomes of the prokaryotes.In an assembling process, due to the secondary structure of various complexity, repetitive sequence
Etc. factors, these data cannot be identified and be correctly assembled on biggish contig during from the beginning assembling, from
And cause a large amount of hole gap.
Summary of the invention
In view of the above problems, the present invention is according to the formation basic theory in this hole, for original high pass amount sequencing data
Assembling result picks up relationship and relevant data between hole gap and contig, provides a kind of utilization prokaryotes base
Because group high quality sketch completes the method for figure.
In order to achieve the above object, The technical solution adopted by the invention is as follows: it is a kind of high-quality using prokaryotic gene group
Amount sketch completes the method for figure, and the method includes the following steps:
1) it is directed to prokaryotic gene group, it is sequenced using high-flux sequence platform, obtains the sketch of high quality；In height
It include the assembling sequence and sequence information of the sequence of each scaffold, the upper each contig of scaffold in quality sketch,
In addition there are also the length estimate in hole etc. is believed between the number of coordinate, read on contig, contig after each read assembling
Breath.
2) in high quality sketch, define contig contig 5 ends ˊ and 3 ends ˊ be node, when contig contig it
Between when there is shared sequence read, then it represents that have side between two nodes, with this basis, established by the principle of graph theory to weigh
Folded group contig and sequence read is the network of content；
3) it according to the sequence for assembling contig contig in result scaffold in sketch, is found out in network different heavy
Existing all shared sequence read between 5 ends ˊ and 3 ends ˊ of folded group contig, are numbered according to read corresponding in sketch
With its coordinate on contig, the corresponding gene order of all shared sequence read is extracted from raw sequencing data
Out, corresponding gene order of all shared sequence read in raw sequencing data is obtained；
4) according to the middle all shared sequence read proposed the corresponding gene sequence in raw sequencing data of step 3)
Column, application sequence editing machine are compiled into the file of fasta format；
5) information in multiple fasta formatted file obtained in step 4) is imported into any a composite software, then
Local assembling is carried out respectively, obtains the part assembling result of multiple corresponding read；
6) for some hole gap on high quality sketch, by the length of hole gap and sequencing depth respectively and in step 5)
The length and sequencing depth of the local assembling result of multiple corresponding read compare, when the part of some read assembles result
It is after length and sequencing depth are identical as the length of hole gap and sequencing depth, the part assembling result of correspondence read is right
Gene order is answered, as the gene order of hole gap on filling high quality sketch, obtains the assembled completion figure in part；
7) operation for repeating step 6), compares all hole gap on high quality sketch respectively, obtains multiple parts
The completion figure of the prokaryotic gene group is made in the assembled unified connection of completion figure in multiple parts by assembled completion figure.
In the high quality sketch that step 2) of the present invention defines, sequence read meets following between contig contig
Condition:
A) when on sequence read 5 ends ˊ and 3 ends ˊ belong to all sequences read of the same contig contig；
B) when on sequence read 5 ends ˊ and 3 ends ˊ be belonging respectively to different contig contig when, sequence read with it is corresponding weight
The length of the lap of folded group contig is necessary >=50bp, the similarity of lap is necessary >=and 90%；
Then determine there is shared sequence read between contig contig.
All shared sequence read corresponding gene orders on sketch must satisfy in step 3) of the present invention:
The Q value > 25 of gene order and the length > 200bp of gene order.
Length of the length and sequencing depth of the part assembling result of read with hole gap in step 6) of the present invention
Criterion identical with sequencing depth: ± the 20% of the length of of length no more than hole gap of the part assembling result of read:
± the 20% of sequencing depth of the sequencing depth no more than hole gap of the part assembling result of read.
The present invention has the advantages that present invention employs the method that bioinformatic data excavates substitute traditional PCR and
Experimental method is sequenced in Sanger.
It is compared with the traditional method, conventional method refers to from the beginning assembling, and what is considered is carried out to whole initial data
Overall situation assembling；In this assembling process, the data such as labyrinth and repetitive sequence cannot be effectively handled, because of this kind of sequence
Generally all repeat repeatedly in whole gene group, and the position of these sequences is difficult to accurately be assembled in real position
It sets.
It is of the present invention and part assembles then there is no problem above, because the complexity within the scope of full-length genome is tied
Structure and repetitive sequence be placed on it is in fact the same with the General Sequences on this region on shorter regional area, so application part
The method of assembling can solve the problem of global assembling can't resolve；The present invention does not need additional experimental cost can save simultaneously
The many experiments time.The result that obtained prokaryotic gene group completion figure and conventional method obtains is just the same.
Detailed description of the invention
Fig. 1 be the present invention is based on E.coli genome original high pass measure sequence sketch, foundation about the end contig
With the network of shared sequence read；
Fig. 2 be the gene completion figure of 5 kinds of bacterial strains from different Pseudomonas that is conventionally measured in the present invention with
The comparing result for the gene order that the present invention measures.
Specific embodiment
The present invention is described in further detail with specific embodiment for explanation with reference to the accompanying drawing.
Embodiment 1: it is as shown in Figure 1 it is a kind of completed the method for figure using prokaryotic gene group high quality sketch,
The method includes the following steps:
1) it is directed to prokaryotic gene group, it is sequenced using high-flux sequence platform, obtains the sketch of high quality；In height
It include the assembling sequence and sequence information of the sequence of each scaffold, the upper each contig of scaffold in quality sketch,
In addition there are also the length estimate in hole etc. is believed between the number of coordinate, read on contig, contig after each read assembling
Breath.
2) in high quality sketch, define contig contig 5 ends ˊ and 3 ends ˊ be node, when contig contig it
Between when there is shared sequence read, then it represents that have side between two nodes, with this basis, established by the principle of graph theory to weigh
Folded group contig and sequence read is the network (as shown in Figure 1) of content；
Wherein sequence read meets the following conditions between contig contig, then determines exist altogether between contig contig
The sequence read enjoyed.
A) when on sequence read 5 ends ˊ and 3 ends ˊ belong to all sequences read of the same contig contig；
B) when on sequence read 5 ends ˊ and 3 ends ˊ be belonging respectively to different contig contig when, sequence read with it is corresponding weight
The length of the lap of folded group contig is necessary >=50bp, the similarity of lap is necessary >=and 90%；
3) it according to the sequence for assembling contig contig in result scaffold in sketch, is found out in network different heavy
Existing all shared sequence read between 5 ends ˊ and 3 ends ˊ of folded group contig, are numbered according to read corresponding in sketch
With its coordinate on contig, the corresponding gene order of all shared sequence read is extracted from raw sequencing data
Out, corresponding gene order of all shared sequence read in raw sequencing data is obtained；All shared sequences
Read corresponding gene order in raw sequencing data must satisfy: the Q value > 25 of gene order and the length of gene order
Spend > 200bp
4) according to the middle all shared sequence read proposed the corresponding gene sequence in raw sequencing data of step 3)
Column, application sequence editing machine: such as editplus software, are compiled into the file of fasta format；
5) information in multiple fasta formatted file obtained in step 4) is imported into any a composite software: such as
Then 7.0 software of DNASTAR carries out local assembling respectively, obtain the part assembling result of multiple corresponding read；
6) for some hole gap on high quality sketch, by the length of hole gap and sequencing depth respectively and in step 5)
The length and sequencing depth of the local assembling result of multiple corresponding read compare, when the part of some read assembles result
It is after length and sequencing depth are identical as the length of hole gap and sequencing depth, the part assembling result of correspondence read is right
Gene order is answered, as the gene order of hole gap on filling high quality sketch, obtains the assembled completion figure in part；
Wherein the length of the part assembling result of read and sequencing depth are identical with the length of hole gap and sequencing depth
Criterion:
A) ± the 20% of the length of of length no more than hole gap of the part assembling result of read:
B) ± the 20% of sequencing depth of the sequencing depth no more than hole gap of the part assembling result of read
7) operation for repeating step 6), compares all hole gap on high quality sketch respectively, obtains multiple parts
The completion figure of the prokaryotic gene group is made in the assembled unified connection of completion figure in multiple parts by assembled completion figure.
Embodiment 2: production method through the invention completes figure to the genome of multiple prokaryotes on the market,
Obtained statistical table is as follows:
1 our company of table completes figure statistical form about the completed genome of prokaryotes
Biggest advantage of the present invention is: on the basis of can guarantee Prokaryotic genome completion figure accuracy, substantially reducing it
Cost of manufacture.
For our company's high-flux sequence cost in 2016 and corresponding experimental cost, table 1 is conventionally obtained
Cited prokaryotic gene group complete figure approximately 100~1,500,000 yuans (according to the complexity of prokaryotic gene group
The estimation of the conditions such as degree, experimental cost, cost of labor): each prokaryotic gene group time needs to expend 3-6 months and differs.
Therefore, the content of all prokaryotes shown in table 1 can not conventionally be completed.
The production that genome completes figure, the totle drilling cost actually spent about 250,000 are carried out according to described in the text method of the invention
Yuan (containing experimental cost and cost of labor), time consumption only 3 months.
Embodiment 3: enterprise conventionally the bacterial strain by wherein 5 plants from different Pseudomonas (including Klebsiella,
Haemophilus parasuis, Streptococcus, Erysipelothrix rhusiopathiae and E.coli) production
Genome completes figure, it is found that the result of conventional method and result of the invention are just the same.
As shown in Fig. 2, sequence alignment result shows the gene order (being measured by conventional method) and other read of Gap1
Gene order is completely the same, and (this comparison result is located at Klebsiella HKOP1 strain gene group 368,950-369,020
It sets).In Fig. 2, with this method obtain hole sequence in addition to conventional method it is completely the same other than, each base information has surveyed 5
Secondary (sequencing depth is 5x), it is therefore contemplated that this method is higher than the accuracy of conventional method.
It should be noted that above-mentioned is only presently preferred embodiments of the present invention, protection model not for the purpose of limiting the invention
It encloses, any combination or equivalents made on the basis of the above embodiments all belong to the scope of protection of the present invention.
Claims (3)
1. a kind of completed the method for figure using prokaryotic gene group high quality sketch, which is characterized in that the method
Include the following steps:
1) it is directed to prokaryotic gene group, it is sequenced using high-flux sequence platform, obtains the sketch of high quality；
2) in high quality sketch, 5 ends ˊ and 3 ends ˊ for defining contig contig are node, are deposited when between contig contig
In shared sequence read, then it represents that have side between two nodes, with this basis, established by the principle of graph theory with contig
Contig and sequence read is the network of content；
3) according to the sequence for assembling contig contig in result scaffold in sketch, different contigs are found out in network
Existing all shared sequence read between 5 ends ˊ and 3 ends ˊ of contig, according to read corresponding in sketch number and its
All shared corresponding gene orders of sequence read are extracted from raw sequencing data, are obtained by the coordinate on contig
Corresponding gene order of all shared sequence read in raw sequencing data out；All shared sequence read are in original
Corresponding gene order must satisfy in beginning sequencing data: the Q value > 25 of gene order and the length > of gene order
200bp；
4) it according to all shared sequence read corresponding gene orders in raw sequencing data proposed in step 3), answers
The file of fasta format is compiled into sequence editor；
5) information in multiple fasta formatted file obtained in step 4) is imported into any a composite software, then distinguished
Local assembling is carried out, the part assembling result of multiple corresponding read is obtained；
6) for some hole gap on high quality sketch, by the length of hole gap and be sequenced depth respectively with it is multiple in step 5)
The length and sequencing depth of the part assembling result of corresponding read compare, when the length of the part assembling result of some read
With sequencing depth with the length of hole gap and sequencing depth it is identical after, by correspondence read part assemble result corresponding to base
Because sequence obtains the assembled completion figure in part as the gene order of hole gap on filling high quality sketch；
7) operation for repeating step 6), compares all hole gap on high quality sketch respectively, obtains multiple part assemblings
The completion figure of the prokaryotic gene group is made in the assembled unified connection of completion figure in multiple parts by good completion figure.
2. according to claim 1 completed the method for figure using prokaryotic gene group high quality sketch, feature
It is, in the high quality sketch that the step 2) defines, sequence read meets following condition between contig contig:
A) when on sequence read 5 ends ˊ and 3 ends ˊ belong to all sequences read of the same contig contig；
B) when on sequence read 5 ends ˊ and 3 ends ˊ be belonging respectively to different contig contig when, sequence read and corresponding contig
The length of the lap of contig is necessary >=50bp, the similarity of lap is necessary >=and 90%；
Then determine there is shared sequence read between contig contig.
3. according to claim 1 completed the method for figure using prokaryotic gene group high quality sketch, feature
It is, the length of the part assembling result of read and sequencing depth are deep with the length of hole gap and sequencing in the step 6)
Identical criterion: ± the 20% of the length of of length no more than hole gap of the part assembling result of read is spent: the office of read
Portion assembles ± the 20% of sequencing depth of the sequencing depth no more than hole gap of result.
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| CN108776749B (en) * | 2018-06-05 | 2022-05-03 | 北京诺禾致源科技股份有限公司 | Sequencing data processing method and device |
| CN111816249B (en) * | 2020-06-01 | 2023-12-08 | 上海派森诺生物科技股份有限公司 | Cyclization analysis method of genome |
Citations (3)
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| CN102272334A (en) * | 2009-01-13 | 2011-12-07 | 关键基因股份有限公司 | Novel genome sequencing strategies |
| CN104531848A (en) * | 2014-12-11 | 2015-04-22 | 杭州和壹基因科技有限公司 | Method and system for assembling genome sequence |
| CN105420375A (en) * | 2015-12-24 | 2016-03-23 | 北京大学 | Method for constructing environmental microbial genome draft |
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- 2016-10-09 CN CN201610879870.XA patent/CN106778060B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102272334A (en) * | 2009-01-13 | 2011-12-07 | 关键基因股份有限公司 | Novel genome sequencing strategies |
| CN104531848A (en) * | 2014-12-11 | 2015-04-22 | 杭州和壹基因科技有限公司 | Method and system for assembling genome sequence |
| CN105420375A (en) * | 2015-12-24 | 2016-03-23 | 北京大学 | Method for constructing environmental microbial genome draft |
Non-Patent Citations (3)
| Title |
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| 6–10× pyrosequencing is a practical approach for whole prokaryote genome studies;Jun Li等;《Gene》;20111213;第494卷(第1期);57-64 |
| A cost-effective and universal strategy for complete prokaryotic genomic sequencing proposed by computer simulation;Jingwei Jiang等;《BMC Reseach Notes》;20120131;第5卷;1-10 |
| High quality draft sequences for prokaryotic genomes using a mix of new sequencing technologies;Jean-Marc Aury等;《BMC Genomics》;20081216;第9卷;1-11 |
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