CN106022002B - A kind of filling-up hole method based on three generations's PacBio sequencing data - Google Patents
A kind of filling-up hole method based on three generations's PacBio sequencing data Download PDFInfo
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Abstract
The invention proposes a kind of filling-up hole methods based on three generations's PacBio sequencing data, greatly reduce the comparison time during filling-up hole, and the speed of genome filling-up hole is improved significantly.Pass through corresponding software, three generations's PacBio sequencing data is compared to the both ends in hole in upper genome, the partial region for three generations's PacBio sequencing data that interception compares, and it is clustered according to data of the hole belonging to the data to interception, error correction is carried out using dazcon software, carries out sequence connection with the data after error correction.
Description
Technical field
The present invention relates to technical field of biological information, and in particular to the filling-up hole method of DNA assembling, it uses three generations PacBio
Sequencing data carries out the filling-up hole of genomic data.
Background technique
Three generations PacBio sequencing is famous with long length of reading, and the P6-C4 reagent used is sequenced at present, can make being averaged for sequencing data
It reads length and reaches 10-15k, and no apparent GC skewed popularity is sequenced, theoretically filling-up hole can be carried out to genome well.At present
Based on the software of three generations's PacBio sequencing data filling-up hole, there is PBjelly, but it is to compare software based on blasr, due to
Blasr software comparison speed is very slow, causes the time for entirely constructing scaffold also very very long.Especially for greater than 1G
Genome, sequencing depth be greater than 10X the case where, it usually needs expend the time of some months.
Summary of the invention
Present invention aim to address posed problems above, propose a kind of benefit based on three generations's PacBio sequencing data
Hole method, greatly reduces the comparison time during filling-up hole, and the speed of genome filling-up hole is improved significantly.By corresponding soft
Three generations's PacBio sequencing data is compared the both ends in hole in upper genome, three generations's PacBio sequencing data that interception compares by part
Partial region, and clustered according to data of the hole belonging to the data to interception, dazcon software used to carry out error correction, used
Data after error correction carry out sequence connection.
The present invention is achieved by the following technical solutions:
The present invention is a kind of filling-up hole method based on three generations's PacBio sequencing data, and the filling-up hole method includes following step
It is rapid:
(1) unique-kmer is extracted from contig；
(2) it uses unique-kmer as seed, is compared, and intercept the region on comparing；
(3) cluster and error correction are carried out to the region in comparison；
(4) it is attached using the data after error correction.
As optimization, k-mer system is carried out to two generation Illumina sequencing datas using Jellyfish software in step (1)
Meter is wrapped, to these unique- using there is primary k-mer as unique-kmer using bit file or GATB open source
Kmer is stored.
K≤17 are stored using the bit file (* .bit file) that a size is 2G as optimization, and for
The case where k > 17, in (* .h5) file in unique-kmer deposit GATB open source packet.
As optimization, the step (2) the following steps are included:
2.1 use unique-kmer as seed；
2.2 in advance cluster the comparison relationship of seed, calculate optimal comparison range；
If two read can be compared, they have synteny, and the slope between these seed is 1,
Using the straight line for gathering most multiple spot as the region on comparing.
2.3 subregions are compared；
The overall region of comparison is divided into the zonule of 100bp first, it is assumed that be divided into n region, share a alkali
Base, then LCS similarity calculation is carried out to these zonules, it is assumed that region of the similarity greater than 0.8 has b, these zonules are total
The similar base of body is c, and following two dimension is divided to evaluate similarity:
Regional Similarity=b/n
Base similarity=c/a
The last value for only retaining two evaluation similarities is both greater than 0.7 comparison data.
As optimization, the step (3) and (4) the following steps are included:
3.1 intercept certain length (can be set as 500bp) is extended before and after the region in comparison, and record this
Hole corresponding to region；
3.2, by the region of interception, are clustered by affiliated hole；
The data of 3.3 pairs of each clusters carry out error correction using dazcon software, then are attached to data.
Compared with existing software, advantage of the process is that
1, unique-kmer is extracted from contig, improves accuracy rate, reduces the reduced time.
In genome, there are many repetitive sequences, some short tandem repeats even occur hundreds and thousands of times, thus can shadow
The accuracy for comparing software is rung, the time of comparison is increased.In order to improve the accuracy of comparison, comparison time is reduced, this law is extracted
Only occur primary k-mer in contig, as unique-kmer, only unique-kmer is used to make as comparison in comparison
Seed.K-mer statistics is carried out used here as Jellyfish software, and filters out unique-kmer.
2, three generations's PacBio sequencing data is compared to the both ends in hole in upper genome, and data intercept, memory is saved, saves
Reduced time improves accuracy.
Many comparison methods all employ the algorithm of longest common subsequence (LCS), and this law is compared, and use
This algorithm, but done the improvement of following three aspect:
1) use unique-kmer as seed
2) the comparison relationship of seed is clustered in advance, calculates optimal comparison range.
If two read can be compared, they have synteny, and the slope between these seed is 1.
We are using the straight line for gathering most multiple spot as the region on comparing.
3) subregion is compared.
Comparing software mostly is all directly to carry out longest common subsequence (LCS) to overall region to calculate, but for larger
Comparison area for, be greater than the comparison area of 100k, overall region calculated, not only wasting memory but also can consume
Take the plenty of time.The improvement that this law is done solves problem above, while accuracy is also significantly improved.
For the three generations's Pacbio sequencing data being compared, the region both ends chosen on comparing respectively extend certain length
The part of (being typically set at 500bp) is intercepted, the hole for ensuring that three generations PacBio sequencing data in this way and comparing
The DNA sequence dna at both ends has common region.
3, three generations's PacBio sequencing data in comparison is clustered, error correction simultaneously connects, and saves the make-up time.
The data of previous step interception are clustered according to respectively affiliated hole, using dazcon software to each hole
Cluster data error correction, and the existing consensus according to data and hole both ends after error correction carry out sequence connection, complete to mend
Hole.Advantage of this is that the regions only to hole to carry out error correction, it is not necessary to error correction is carried out to whole sequence, to be saved greatly
Make-up time.
Detailed description of the invention
Fig. 1: flow chart of the present invention.
Specific embodiment
The embodiment of the present invention is further elaborated with reference to the accompanying drawing:
Embodiment:
1, unique-kmer is extracted from contig, uses Jellyfish software to two generation Illumina in step (1)
Sequencing data carries out k-mer statistics, and the k-mer for occurring primary is used a size for k≤17 as unique-kmer
It is stored for the bit file (* .bit file) of 2G, and the case where for k > 17, unique-kmer deposit GATB open source packet
In (* .h5) file in.Wherein, all data are broken into the segment that length is k and are known as k-mer, two generations Illumina surveyed
Ordinal number is according to the two generation sequencing datas referred to through the acquisition of Illumina company sequenator.
Program is write according to the above method, usage is as follows:
The path contig is put into one file f ile.lst
Then program is run, to obtain unique-kmer:
Because choosing k=17, result is stored in bit file: k17.bit
2, use unique-kmer as seed, be compared, and intercept the region on comparing；
2.1 use unique-kmer as seed；
2.2 in advance cluster the comparison relationship of seed, calculate optimal comparison range；
If two read can be compared, they have synteny, and the slope between these seed is 1,
Using the straight line for gathering most multiple spot as the region on comparing.
2.3 subregions are compared.
The overall region of comparison is divided into the zonule of 100bp first, it is assumed that be divided into n region, share a alkali
Base, then LCS similarity calculation is carried out to these zonules, it is assumed that region of the similarity greater than 0.8 has b, these zonules are total
The similar base of body is c, and following two dimension is divided to evaluate similarity:
Regional Similarity=b/n
Base similarity=c/a
The last value for only retaining two evaluation similarities is both greater than 0.7 comparison data.
3, cluster and error correction are carried out to the region in comparison, is attached using the data after error correction.
3.1 intercept certain length (can be set as 500bp) is extended before and after the region in comparison, and record this
Hole corresponding to region；
3.2, by the region of interception, are clustered by affiliated hole；
The data of 3.3 pairs of each clusters carry out error correction using dazcon software, then are attached to data.
According to the method for above-mentioned comparison and filling-up hole, says that comparison and filling-up hole method are write as a process, facilitate calling, usage is such as
Under:
Prepare the explanation of input.cfg:
What has been described above is only a preferred embodiment of the present invention, it is noted that for common skill in the art
For art personnel, under the premise of not departing from core technical features of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (4)
1. a kind of filling-up hole method based on three generations's PacBio sequencing data, which is characterized in that the filling-up hole method includes following step
It is rapid:
(1) unique-kmer is extracted from contig；
(2) it uses unique-kmer as seed, is compared, and intercept the region on comparing, comprising the following steps:
2.1 use unique-kmer as seed；
2.2 in advance cluster the comparison relationship of seed, calculate optimal comparison range；
If two read can be compared, they have synteny, and the slope between these seed is 1, will be gathered
To most multiple spot straight line as compare on region；
2.3 subregions are compared；
The overall region of comparison is divided into the zonule of 100bp first, it is assumed that it is divided into n region, shares a base, then
LCS similarity calculation is carried out to these zonules, it is assumed that region of the similarity greater than 0.8 there are b, the phase of these zonules totality
It is c like base, following two dimension is divided to evaluate similarity:
Regional Similarity=b/n；
Base similarity=c/a；
The last value for only retaining two evaluation similarities is both greater than 0.7 comparison data；
(3) cluster and error correction are carried out to the region in comparison；
(4) it is attached using the data after error correction.
2. the filling-up hole method according to claim 1 based on three generations's PacBio sequencing data, which is characterized in that in step
(1) k-mer statistics is carried out to two generation Illumina sequencing datas using Jellyfish software, using occur primary k-mer as
Unique-kmer stores these unique-kmer using bit file or GATB open source packet.
3. the filling-up hole method according to claim 2 based on three generations's PacBio sequencing data, which is characterized in that for k
≤ 17, it is stored using the bit file * .bit that size is 2G, and the case where for k > 17, unique-kmer is deposited
Enter in the * .h5 file in GATB open source packet.
4. the filling-up hole method according to claim 1 based on three generations's PacBio sequencing data, which is characterized in that the step
Suddenly (3) and (4) the following steps are included:
3.1 intercept certain length is extended before and after the region in comparison, and record hole corresponding to this region；
3.2, by the region of interception, are clustered by affiliated hole；
The data of 3.3 pairs of each clusters carry out error correction using dazcon software, then are attached to data.
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| CN106599617B (en) * | 2016-12-20 | 2019-02-15 | 福建师范大学 | A kind of magnanimity sequencing data error correcting method running on distributed system |
| CN108629156B (en) * | 2017-03-21 | 2020-08-28 | 深圳华大基因科技服务有限公司 | Method, device and computer readable storage medium for correcting error of third generation sequencing data |
| CN107256335A (en) * | 2017-06-02 | 2017-10-17 | 肖传乐 | A kind of preferred three generations's sequencing sequence comparison method of being given a mark based on global seed |
| CN107229842A (en) * | 2017-06-02 | 2017-10-03 | 肖传乐 | A kind of three generations's sequencing sequence bearing calibration based on Local map |
| CN108763871B (en) * | 2018-06-05 | 2022-05-31 | 北京诺禾致源科技股份有限公司 | Hole filling method and device based on third-generation sequencing sequence |
| CN109411020B (en) * | 2018-11-01 | 2022-02-11 | 中国水产科学研究院 | Method for hole filling of whole genome sequence by using long sequencing reads |
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| 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 |
| CN104951672B (en) * | 2015-06-19 | 2017-08-29 | 中国科学院计算技术研究所 | Joining method and system associated with a kind of second generation, three generations's gene order-checking data |
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