CN107841542A - A kind of generation sequence assemble method of genome contig two and system - Google Patents
A kind of generation sequence assemble method of genome contig two and system Download PDFInfo
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Abstract
The invention discloses a kind of generation sequence assemble method of genome contig two and system, methods described to include：Sample gene group DNA is interrupted to the first predetermined length scope；Glue is cut to the DNA fragmentation interrupted and selects the second predetermined length scope, to build the library of different Insert Fragments；Double end sequencings are carried out to the DNA fragmentation of the second predetermined length scope, obtain that there is overlapping first to read long sequence and the second long sequence of reading for specific DNA fragments；First obtained to the double end sequencings in each library reads long sequence and the second long sequence of reading is spliced, and obtains the spliced sequence in each library；Sequence assembling is carried out to the spliced sequence in each library to obtain genome Contig.The method of the present invention builds storehouse by experiment and sequencing reading length is chosen, and combines splicing to realize that extending sequence reads length, is finally assembled with the sequence after extending, obtains contig, it is possible to increase the index and accuracy of genome contig assembling.
Description
Technical field
The present invention relates to gene sequencing technology field, more particularly to a kind of generation sequence assemble method of genome contig two and
System.
Background technology
At present, (Whole-genome shotgun are sequenced with whole-genome shotgun sequencing in genome assembling project
Sequencing, WGS) it is main flow design, it is mainly according to the specific feature of the repetitive sequence of genome, different length of arranging in pairs or groups
The DNA Insert Fragments of degree carry out double end sequencings, can ensure single alkali in the case where the average sequencing depth of full-length genome is enough
The accuracy of base and the integrality of genome.With second generation sequencing technologies (Next-generation sequencing, NGS)
Maturation and popularization, sequencing cost substantially reduce, the sequencing of whole-genome shotgun sequencing based on second generation sequencing technologies turns into various
The mainstream scheme of Genome Project sequencing.And the contig that whether can assemble a high quality is often related to genome skeleton
One important factor in order of sequence assembling effect quality.
The contig assembled is arranged from big to small, when its cumulative length is just beyond all assembling sequence total lengths
When 50%, the size of last contig (Contig) is N50 size, and N50 has to the integrality for evaluating gene sequencing
Significance.N60 from big to small arranges the contig assembled, when its cumulative length is total just beyond all assembling sequences
During length 60%, the size of last contig (Contig) is N60 size.N70, N80, N90 are by that analogy.
The content of the invention
Sequence reads long (reads) and genome assembling effect is had a major impact, and method and system of the invention passes through experiment
Build storehouse and sequencing reading length is chosen, and combine splicing to realize that extending sequence reads length, is finally assembled with the sequence after extending, obtained
To contig, it is possible to increase the index and accuracy of genome contig assembling.
According to the first aspect of the invention, the present invention provides a kind of generation sequence assemble method of genome contig two, including：
Sample gene group DNA is interrupted to the first predetermined length scope；Glue is cut to the DNA fragmentation interrupted and selects the second predetermined length model
Enclose, to build the library of different Insert Fragments；Double end sequencings, pin are carried out to the DNA fragmentation of above-mentioned second predetermined length scope
Specific DNA fragments are obtained with there is overlapping first to read long sequence and the second long sequence of reading；The double end sequencings in each library are obtained
To first read long sequence and second and read long sequence and spliced, obtain the spliced sequence in each library；Each library is spelled
Sequence after connecing carries out sequence assembling to obtain genome Contig.
Further, above-mentioned sample gene group DNA is interrupted using ultrasound.
Further, above-mentioned first predetermined length scope is 100bp-600bp or 100bp-500bp.
Further, above-mentioned second predetermined length scope be 170bp-180bp, 260bp-280bp, 450bp-470bp or
550bp-570bp。
Further, the long sequence of above-mentioned first reading and the second sequence length for reading long sequence are 100bp-300bp.
Further, the long sequence of above-mentioned first reading and the second sequence length for reading long sequence are 100bp, 150bp, 250bp
Or 300bp.
Further, it is above-mentioned to be specifically to the spliced sequence in each library progress sequence assembling：By two generation sequencing sequences
The short sequence K-mer that length is K is intercepted out successively；By K-mer storages into hash table, the summit of de Brujin graph is formed；
The K-mer of successive is connected on sequencing sequence, forms the side of de Brujin graph；All sequencing sequences are all handled to obtain whole
Individual de Brujin graph；Remove the path as caused by sequencing mistake, heterozygous sites in de Brujin graph；By linear K-mer paths
Connect the contig to form the first order.
Further, above-mentioned short sequence K-mer length is 30bp-500bp.
Further, the above method also includes：Before above-mentioned splicing, sequence containing joint and low is removed by filtering off
Mass-sequential.
According to the second aspect of the invention, the present invention provides a kind of generation sequence package system of genome contig two, including：
Module is interrupted, for sample gene group DNA to be interrupted to the first predetermined length scope；Selecting module, for the DNA pieces to interrupting
Section cuts glue and selects the second predetermined length scope, to build the library of different Insert Fragments；Sequencer module, for pre- to above-mentioned second
The DNA fragmentation of measured length scope carries out double end sequencings, for specific DNA fragments obtain having overlapping first read long sequence and
Second reads long sequence；Concatenation module, first for being obtained to the double end sequencings in each library, which reads long sequence and second, reads long sequence
Row are spliced, and obtain the spliced sequence in each library；Module is assembled, for carrying out sequence to the spliced sequence in each library
Row are assembled to obtain genome Contig.
The method and system of the present invention builds storehouse by experiment and sequencing reading length is chosen, and combines splicing to realize extension sequence
Length is read, is finally assembled with the sequence after extending, obtains contig, it is possible to increase the index and standard of genome contig assembling
True property.
Brief description of the drawings
Fig. 1 shows one embodiment flow chart of the generation sequence assemble method of genome contig two of the present invention；
Fig. 2 shows that the present invention cuts the DNA molecular after glue and reads long sequence and second using first and reads long sequence and survey to lead to and carry out
Splicing, obtain one embodiment flow chart of longer sequence；
Fig. 3 shows one embodiment structured flowchart of the generation sequence package system of genome contig two of the present invention.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.
In one embodiment of the invention, there is provided one kind is built storehouse, cut glue technology based on second generation sequencing technologies and experiment
It is combined, it is intended to improve the method and system of genome contig assembling effect.
Fig. 1 shows that the present invention builds storehouse based on second generation sequencing technologies and experiment, cuts glue technology and be combined, and assembles genome weight
One embodiment flow chart of folded group.
As shown in figure 1, in a step 102, reading to grow with reference to two generation sequencing sequences, sample progress DNA is interrupted some
Length range (i.e. the first predetermined length scope).In one embodiment of the invention, sample gene group DNA is beaten using ultrasound
Disconnected, the first predetermined length scope is 100bp-600bp or 100bp-500bp.
At step 104, the sequence of the double end sequencings of selection reads length as needed, it is determined that cutting the length range (i.e. the of glue
Two predetermined length scopes), corresponding example in one embodiment is given in table 1.
Table 1
In step 106, the length range of glue is cut according to step 104, corresponding length of reading is chosen and carries out double end sequencings, and protect
Card read 1 and read 2 (i.e. first, which reads long sequence and second, reads long sequences) have it is overlapping, as given in table 1 in one embodiment accordingly
Example.
In step 108, after step 106 obtains sequence, it is compared to reading 1 end and reading 2 front ends, if on comparing
Just spliced, obtain spliced sequence.This step can use PEAR alignment and assembbly softwares, can be from http://sco.h-
Its.org/exelixis/web/software/pear/ is obtained.
In step 110, the splicing sequence obtained with step 108 is assembled, and obtains Contig.By two generation sequences
Assembled, long (sequencing sequence) will be read and intercept out the short sequence that length is K successively, weighed mutually before and after referred to as K-mer, K-mer
Fold K-1 base.By K-mer storages into hash table, the summit of de Brujin graph is formed；The K-mer of successive in reading
Think that the two K-mer are connected, form the side of de Brujin graph.After all reading length have all been handled, whole moral cloth can be obtained
Lu Yintu, the path as caused by sequencing mistake, heterozygous sites in figure is removed, linear K-mer paths are connected can shape
Into Contig (contig) sequence of the first order.These K-mer bases are connected to the Contig for forming the first order.
The assembling in this stage can use splicing software SOAPdenovo or Platanus.SOAPdenovo composite software bibliography
Li,R.et al.De novo assembly of human genomes with massively parallel short
read sequencing.Genome Res(2009).This software can be obtained freely from network, network address http://
soap.genomics.org.cn/soapdenovo.html.Or can http from network://
Platanus.bio.titech.ac.jp/platanus/ obtains Platanus composite softwares.
Fig. 2 shows that the present invention builds storehouse based on second generation sequencing technologies and experiment, cuts glue technology and be combined, and obtains longer sequence
Read long one embodiment flow chart.
In step 202, show by cutting the DNA molecular obtained after glue.
In step 204, show with reference to glue scope is cut, choose corresponding sequencing reading length technology, obtaining reading 1 and reading 2 has weight
The sequence in folded region.
In step 206, the sequence after reading 1 and 2 alignment and assembblies of reading is shown.
Corresponding to the generation sequence assemble method of genome contig two shown in Fig. 1, the present invention also provides a kind of genome weight
The folded generation sequence package system of group two, as shown in figure 3, including：Module 310 is interrupted, for sample gene group DNA to be interrupted to first
Predetermined length scope；Selecting module 320, the second predetermined length scope is selected for cutting glue to the DNA fragmentation interrupted, to build not
With the library of Insert Fragment；Sequencer module 330, surveyed for carrying out double ends to the DNA fragmentation of above-mentioned second predetermined length scope
Sequence, obtain that there is overlapping first to read long sequence and the second long sequence of reading for specific DNA fragments；Concatenation module 340, for pair
The long sequence of the first reading and the second long sequence of reading that the double end sequencings in each library obtain are spliced, after obtaining each library splicing
Sequence；Module 350 is assembled, for carrying out sequence assembling to the spliced sequence in each library to obtain genome contig sequence
Row.
The algae bryophyte Genome Size that the inventive method is provided below is about 400MB concrete application examples.In the example
In son, the sequencing assembling of genome contig is realized, is comprised the following steps that：
(1) storehouse sequencing is built
Extract the DNA of sample and interrupt at random, after electrophoresis, cut 170bp-180bp and 250bp-260bp scopes respectively
Gel-purified.DNA fragmentation connection sequence measuring joints after purification, PCR amplifications, then carry out double ends using two generation sequenators respectively
100bp and 150bp sequences are held to read long sequencing.
(2) data filtering
Some original series carry joint sequence, or contain a small amount of low quality sequence.We pass through one first by software
Series data processing obtains valid data to remove impurity data.Filtration step specifically includes：
1) sequence containing joint is removed；
2) remove low quality sequence (base number of the mass value less than or equal to 20 accounts for more than the 20% of whole sequence)；
3) sequence after being filtered.
(3) sequence assembly
Splicing is compared to the sequence after filtering respectively by PEAR softwares, obtains spliced sequence.
(4) contig is established.
Sequence after splicing is assembled with Platanus softwares, obtains the Contig that size is about 419Mb.
Contig N50 are 1881bp.Table 2 is reading 1 and the splicing of reading 2, does not splice the assembling effect comparison sheet being compared.Can from table
To find out reading 1 and read 2 splicings than not splicing progress contig assembling, assembling result N50, N60, N70, N80, which are improved, to be exceeded
100%, N90 also improve 59%.
Table 2
| Parameter | Read 1 and read 2 splicings (bp) | Read 1 and do not splice (bp) with reading 2 |
| Contig N50 | 2074 | 607 |
| Contig N60 | 947 | 313 |
| Contig N70 | 466 | 205 |
| Contig N80 | 315 | 137 |
| Contig N90 | 174 | 109 |
Above content is to combine specific embodiment further description made for the present invention, it is impossible to assert this hair
Bright specific implementation is confined to these explanations.For general technical staff of the technical field of the invention, do not taking off
On the premise of from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the protection of the present invention
Scope.
Claims (10)
1. a kind of generation sequence assemble method of genome contig two, it is characterised in that methods described includes：
Sample gene group DNA is interrupted to the first predetermined length scope；
Glue is cut to the DNA fragmentation interrupted and selects the second predetermined length scope, to build the library of different Insert Fragments；
Double end sequencings are carried out to the DNA fragmentation of the second predetermined length scope, obtain having for specific DNA fragments overlapping
First read long sequence and second and read long sequence；
First obtained to the double end sequencings in each library reads long sequence and the second long sequence of reading is spliced, and obtains each library
Spliced sequence；
Sequence assembling is carried out to the spliced sequence in each library to obtain genome Contig.
2. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that the sample gene
Group DNA is interrupted using ultrasound.
3. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that described first is predetermined
Length range is 100bp-600bp or 100bp-500bp.
4. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that described second is predetermined
Length range is 170bp-180bp, 260bp-280bp, 450bp-470bp or 550bp-570bp.
5. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that described first reads length
Sequence and the second sequence length for reading long sequence are 100bp-300bp.
6. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that described first reads length
Sequence and the second sequence length for reading long sequence are 100bp, 150bp, 250bp or 300bp.
7. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that described to each text
The spliced sequence in storehouse carries out sequence assembling：Two generation sequencing sequences are intercepted into out the short sequence K- that length is K successively
mer；By K-mer storages into hash table, the summit of de Brujin graph is formed；The K-mer phases of successive on sequencing sequence
Even, the side of de Brujin graph is formed；All sequencing sequences have all been handled to obtain whole de Brujin graph；Remove de Brujin graph
In as sequencing mistake, path caused by heterozygous sites；Linear K-mer paths are connected to the contig to form the first order.
8. the generation sequence assemble method of genome contig two according to claim 7, it is characterised in that the short sequence K-
Mer length is 30bp-500bp.
9. the generation sequence assemble method of genome contig two according to claim 1, it is characterised in that methods described is also wrapped
Include：Before the splicing, sequence and low quality sequence containing joint are removed by filtering off.
10. a kind of generation sequence package system of genome contig two, it is characterised in that the system includes：
Module is interrupted, for sample gene group DNA to be interrupted to the first predetermined length scope；
Selecting module, the second predetermined length scope is selected for cutting glue to the DNA fragmentation interrupted, to build different Insert Fragments
Library；
Sequencer module, for carrying out double end sequencings to the DNA fragmentation of the second predetermined length scope, for specific DNA pieces
Section obtains that there is overlapping first to read long sequence and the second long sequence of reading；
Concatenation module, read long sequence for the long sequence of the first reading obtained to the double end sequencings in each library and second and spell
Connect, obtain the spliced sequence in each library；
Module is assembled, for carrying out sequence assembling to the spliced sequence in each library to obtain genome Contig.
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