CN107895104A - Assess and verify the method and apparatus of the sequence assembling result of three generations's sequencing - Google Patents
Assess and verify the method and apparatus of the sequence assembling result of three generations's sequencing Download PDFInfo
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Abstract
The invention discloses a kind of method and apparatus for the sequence assembling result for assessing and verifying three generations's sequencing.The method of the sequence assembling result provided by the present invention for assessing three generations's sequencing includes：Two generation sequences assemble result with three generations and compared；Low cover degree region extends and chosen, sequence after being extended；Three generation sequences compare with extension sequence；Base overburden depth counts；Assemble result queue.It is not too high region that can filter out three generations to assemble quality in result by the present invention, and is marked out and.In follow-up species research, there is the function of prompting in the region for using these of low quality if desired, and provides quick screening means for follow-up improvement.It also can prove that three generations assembles the accuracy and quality of result simultaneously, the accuracy of assembling result can be improved.
Description
Technical field
The invention belongs to gene order-checking field, is related to a kind of side for the sequence assembling result for assessing and verifying three generations's sequencing
Method and device.
Background technology
Contig (contig) is by being assembled into the splicing of overlapping (overlap) region without between by sequence (reads)
The tract of gap (gap)；The contig (contig) that frame sequence (scaffold) is determined by double terminal position information is arranged
Row, there is gap centre.The contigs or scaffolds that assemble are arranged from big to small, when its cumulative length is just beyond complete
When portion assembles sequence total length 50%, last contig or scaffold size are N50 size, and N50 is to evaluation
Continuity, the integrality for assembling sequence are significant；N70 and N90 computational methods are similar with N50, and simply percentage is changed into
70% or 90%.
The sequencing of two generations due to reading long limitation (generally 50bp-300bp), using splicing two kinds of algorithms OLC and DBG all
Can not be across long repeat region, in assembling running into these repeat regions can all be disconnected.Although difference can be used
Gradient large fragment (such as 2k, 5k, 10k, 20k, 40k etc.) data connect two contigs using the comparison relation of double terminal positions
Pick up to be spliced into frame sequence (Scaffold), but Contig N50 length is not still long (generally 1k-70k).
(SMRT) technology is sequenced due to reading long (averagely read long general with overlength in three generations's sequencing-Pacbio unimolecules in real time
In 8k-13k) the characteristics of, the genome complex regions such as high repetitive sequence, swivel base subregion and height variable region can be carried out high
Level assembling so that contig (Contig) N50 and frame sequence (Scaffold) N50 length are longer, and assembling result is more complete
Accurately, as three generations is sequenced, cost is more and more lower, and it is also more and more that three generations assembles Genome Project.Three generations's composite software master at present
There are PBCR, Falcon, MECAT, CANU, HGAP etc., these softwares all include the work(from sequence self assembly after error correction and error correction
Energy.Because three generations's sequence average error rate is up to 15%, therefore these softwares are required for first carrying out self-picketing mistake, recycle error correction postorder
Row are assembled, and finally obtain assembling result, because assembling result there may be certain single base mistake or structure variation, institute
Subsequently to need to carry out Polish error correction with three generations's original series, and Pilon error correction is carried out with two generation sequences, obtain final three
Generation assembling result, the main process of three generations's assembling are as shown in Figure 1.
After obtaining assembling result, we can be evaluated the quality for assembling result by different methods.Such as：
(1) utilize same individual BAC/Fosmid sequences (or BAC/Fosmid sequences of species of the same race), by with genome
Sequence alignment, genome euchromatin coverage is examined, as shown in Fig. 2 being one section of Fosmid sequence above, here is for we
Assemble result sequence, they compare effect it is very good, it was demonstrated that this section of Fosmid sequence be assembled out and effect very
It is good.(2) existing est sequence is utilized, by being compared with genome sequence, examines the coverage of gene regions.(3) single base is covered
Lid depth assessment, two generation sequences compare the overburden depth for assembling result to three generations and counting three generations's assembling each base of result.Such as
Shown in Fig. 3, the average overburden depth 80X of two generation sequences, X-axis represents the overburden depth in different sections, and Y-axis represents different sections and covered
Ratio corresponding to lid depth, from the point of view of this figure, the ratio less than 10X overburden depths is lower, the group of reaction assembling result single base
Higher (four) the G/C content distributional analysis of packing quality.As shown in figure 4, abscissa is G/C content, ordinate is mean depth.Two article used in lieu of a preface
Row, which compare, to be assembled result to three generations and counts the overburden depth that three generations assembles each base of result, is entered using 10kb as window without repetition
Row calculates.According to this figure, we can analyze the G/C content of this species, whether can have to the sample exogenous DNA pollute into
Row judges.In addition it is also seen that the assembling quality effect of our subregions.Fig. 4 (B) result display assembling result G/C content
Depth profile is normal, but shown in Fig. 4 (A), there is part low depth overlay area, causes the possibility of this phenomenon and has two reasons, and one
It is that three generation sequences are relatively low in these region overlay depth, causes assembling result certain base mistake or missing to be present, although through
Three generations polish error correction and two generation pilon error correction are crossed, but correction does not come；Second, the assembling of this subregion is accurate,
Region overlay depth is very high in this section by three generations, but two generations, region overlay depth was relatively low in this section, may be due to mistake is sequenced
Cause to compare less than this subregion, or the part that this subregion does not measure or measured is relatively low.
The content of the invention
In order to effectively solve three generations assemble result overburden depth is relatively low in two generation sequences for subregion is which kind of reason
Caused, the invention provides a kind of method and apparatus for the sequence assembling result for assessing and verifying three generations's sequencing.
The method of the sequence assembling result provided by the present invention for assessing three generations's sequencing, generally comprises following steps：
(1) the sequence assembling result that two generation sequencing sequences of same sample are sequenced with three generations is contrasted.
(2) according to the comparison result of step (1), picked out from the sequence assembling result of three generations sequencing described two
For the low region of average overburden depth in sequencing sequence, then by each selected region in the sequence of three generations sequencing
Extended in assembling result, so as to obtain sequence after several extensions.
(3) sequence is individually compared after each extension for obtaining three generations's sequencing sequence with step (2).
(4) according to the comparison result of step (3), (i.e. in each two generation, is low in each selected region in statistic procedure (2)
Overburden depth region) average overburden depth in three generations's sequencing sequence.
(5) according to the statistical result of step (4), the height of the assembling quality in each region selected in step (2) is determined
It is low, and then realize the assessment of the sequence assembling result to three generations sequencing.
Specifically, methods described comprises the following steps：
(1) being contrasted the sequence assembling result that two generation sequencing sequences of same sample are sequenced with three generations (can use
The softwares such as software bwa or SOAPAligner are compared to complete), count each base in the sequence assembling result of three generations's sequencing
Overburden depth (can be carried out using SOAPCoverage softwares) in the two generations sequencing sequence, so it is (specific with 1-5kb
Such as 1kb) it is window, each window area in the sequence assembling result of three generations's sequencing is calculated sequence is sequenced in two generation
Average overburden depth in row.
(2) according to the result of step (1), pick out from the sequence assembling result of three generations sequencing and surveyed in two generation
The low whole window areas of average overburden depth in sequence sequence, then survey each selected window area in the three generations
Respectively extend 10-40kb (specific such as 30kb) in the sequence assembling result of sequence forwards, backwards, so as to obtain sequence after several extensions.
(3) sequence carries out individually comparing (comparison software after each extension for obtaining three generations's sequencing sequence and step (2)
Bwa can be used).
(4) according to the comparison result of step (3), selected each window area (i.e. former 1-5kb in statistic procedure (2)
Two generations low overburden depth region) average overburden depth in three generations's sequencing sequence.
(5) according to the statistical result of step (4), according to (i.e. former to each window area selected in step (2) as follows
The generations of 1-5kb bis- low overburden depth region) carry out the mark of assembling quality height, and then assess the sequence assembling of three generations sequencing
As a result overall package quality：If selected some window area A is flat in three generations's sequencing sequence in step (2)
Equal overburden depth is less than or equal to 5X, then the window area A is labeled as into " the relatively low region of assembling quality "；If step
(2) average overburden depths of selected some window area B in three generations's sequencing sequence is more than 5X in, then by the window
Mouth region domain B is labeled as " the of a relatively high region of assembling quality ".
The methods described the step of in (5), specifically according to the sequence assembling result for assessing three generations's sequencing as follows
Overall package quality：The quantity in " the of a relatively high region of assembling quality " that is marked with described " assembling quality is relative
The ratio of both relatively low region " and " the of a relatively high region of assembling quality " total quantity is bigger, then three generations's sequencing
Sequence assembling result overall package quality it is higher.
Wherein, if continuous 2 or multiple while being marked as " assembling quality in window area selected in step (2)
Relatively low region ", then they are merged into note into one " the relatively low region of assembling quality "；If continuous 2 or more
Individual while be marked as " the of a relatively high region of assembling quality ", then they being merged into note, " assembling quality is of a relatively high into one
Region ".
The methods described the step of in (1), the two generations sequencing sequence is the initial data obtained by two generation high-flux sequences
Sequence (eliminating joint and low quality base) after filtration treatment.In one embodiment of the invention, the sample
This is Maize genome, and the two generations sequencing sequence is specially that the HiSeq2500 platforms 250PE sequencing gained of Maize genome is former
Beginning sequence filter falls the part after joint and low quality base.
Further, needed in the present invention by initial data to valid data filtering by three step process：
1) transition joint：Sequencing read matches the 50% of adapter sequences or then deletes whole piece reads above；
2) low quality data is filtered：If base of the mass value less than 20 accounts for whole piece read 10%- in sequencing read
Whole piece read is deleted if 50% (specific such as 20%) or the above；
3) N is removed：If in sequencing reads N content account for whole piece read 1%-10% (specific as 2%) or more than,
Delete whole piece read.Wherein, N represents the base that sequencing is not measured.
The methods described the step of in (3), three generations's sequencing sequence is non-error correction sequence or from after error correction
Sequence.Wherein, the non-error correction sequence of three generations has two kinds, and a kind of is the subreads sequences that PacBio RSII machine sequencings obtain, separately
A kind of is the sequence that the bam formatted datas that Sequal machine sequencings obtain are converted into fasta forms；Refer to from the sequence after error correction
It is the sequence that three generations's initial data obtains from after error correction.In one embodiment of the invention, the sample is Maize genome,
Three generations's sequencing sequence is specially that original series process obtained by (SMRT) is sequenced in the Pacbio unimolecules of Maize genome in real time
From the sequence after error correction.
The methods described the step of in (2), the average overburden depth is low to refer to that average overburden depth is less than " low depth
Define threshold values ", it is following any：
(a1) when the average overburden depth of two generation sequencing is 30X, described " low depth defines threshold values " is 3X.
(a2) when the average overburden depth of two generation sequencing is more than 30X and is less than or equal to 50X, described " low depth is fixed
Adopted threshold values " is 4-5X.
(a3) when the average overburden depth of two generation sequencing is more than 50X and is less than or equal to 70X, described " low depth is fixed
Adopted threshold values " is 6-8X.
(a4) when the average overburden depth of two generation sequencing is more than 70X, described " low depth defines threshold values " is 9-
10X。
In one embodiment of the invention, the sample is Maize genome, and the average covering of the two generations sequencing is deep
It is 6X to spend for 60X, described " low depth defines threshold values ".
The methods described the step of in (2), described " by each selected window area, (in i.e. former generations of 1kb bis-, low covering was deep
Spend region) respectively extend 30kb " forwards, backwards in the sequence assembling result of three generations sequencing, if selected window area is in its institute
Frame sequence (Scaffold) in, less than 30kb before or after it, then extend to existing part and terminate.I.e.：Such as
Fruit respectively extends 30kb forwards, backwards, obtains the sequence after extension, and each back zone length of field that extends amounts to 61kb；Before and after Scaffold such as
Fruit is less than 30k, the part taken, for example window is 5-6k regions, only takes 0-5k forward, removes 6-36k, i.e. 1-36kb backward；
It is insufficient behind Scaffold, get last base from window end.Because the length range of three generation sequences is hundreds of bp
To tens kb, and these low depth overlay areas may as little as tens bp, so the present invention is to each before and after low depth overlay area
30kb is extended, to ensure that three generation sequences can be compared with these regions.
The methods described the step of in (1), the average amount of the two generations sequencing reach the 30X of Genome Size with
On, preferably more than 50X.In one embodiment of the invention, the sample is Maize genome, and the two generations sequencing is put down
Equal data measurer body is the 60X of Maize genome size.
The methods described the step of in (1), the base mismatch number allow during the comparison is more preferably less than equal to 2.
In the process, three generations's sequencing is sequenced (SMRT) in real time for Pacbio unimolecules；Three generations's sequencing
Average amount is preferably in more than the 50X of Genome Size.In one embodiment of the invention, the sample is corn gene
Group, the average amount of three generations's sequencing is specially the 80X of Maize genome size.
The assessment system (device) of the sequence assembling result of three generations's sequencing provided by the present invention, including data processing equipment
A, data processing equipment B, data processing equipment C, data processing equipment D and data processing equipment E.
Module a1, module a2, module a3 are set in the data processing equipment A；Sequence can be sequenced to two generations in the module a1
Arrange and contrasted with the sequence assembling result of three generations's sequencing；The result that the module a2 can compare according to the module a1, system
Count each overburden depth of the base in the two generations sequencing sequence in the sequence assembling result of three generations's sequencing；The module
According to the statistical result of the module a2, with 1-5kb (specific such as 1kb) for window, three generations's sequencing can be calculated in a3
Sequence assembling result in each average overburden depth of the window area in the two generations sequencing sequence.
In the present invention, the module a1 is specially bwa softwares or SOAPAligner softwares；The module a2 is specially
SOAPCoverage softwares.
Module b1 and module b2 are set in the data processing equipment B；The module b1 can fill according to the data processing
The result of A acquisitions is put, picks out from the sequence assembling result of three generations sequencing and is averagely covered in the two generations sequencing sequence
The low whole window areas of lid depth；The module b2 can be by each window area selected by the module b1 described
Respectively extend 10-40kb (specific such as 30kb) in the sequence assembling result of three generations's sequencing forwards, backwards, after obtaining several extensions
Sequence.
Module c1 is set in the data processing equipment C；The module c1 can be by three generations's sequencing sequence and the data
Sequence is individually compared after each extension that reason device B is obtained.
In the present invention, the module c1 is specially bwa softwares.
Module d1 is set in the data processing equipment D；What the module d1 can obtain according to the data processing equipment C
Comparison result, (in the i.e. former generations of 1kb bis-, are low for each window area for counting described in the data processing equipment B selected by module b1
Overburden depth region) average overburden depth in three generations's sequencing sequence.
Module e1 and module e2 are set in the data processing equipment E；The module e1 can fill according to the data processing
The statistical result of D acquisitions is put, according to as follows to each window area selected by module b1 described in the data processing equipment B
(i.e. former the generations of 1kb bis- low overburden depth region) carries out the mark of assembling quality height：If some window area A is described three
It is less than or equal to 5X for the average overburden depth in sequencing sequence, then by the window area A labeled as " assembling quality is relatively low
Region "；If average overburden depths of some window area B in three generations's sequencing sequence is more than 5X, by described in
Window area B is labeled as " the of a relatively high region of assembling quality "；The module e2 can be according to the mark knot of the module e1
Fruit statistics calculates " the of a relatively high region of assembling quality " that module b1 is marked described in the data processing equipment B
Quantity and both " the relatively low region of assembling quality " and " the of a relatively high region of assembling quality " total quantity
Ratio；Wherein, if continuous 2 or multiple while being marked as that " assembling quality is relative in window area selected by the module b1
Relatively low region ", then they are merged into note into one " the relatively low region of assembling quality "；If continuous 2 or multiple same
When be marked as " the of a relatively high region of assembling quality ", then they are merged into note into " of a relatively high an area of assembling quality
Domain ".
Methods described or the system it is following it is any in application fall within protection scope of the present invention：
(A) the sequence assembling result of three generations's sequencing is assessed；
(B) screen and mark ropy region in the sequence assembling result that three generations is sequenced.
It is demonstrated experimentally that using assessment provided by the present invention and the method for the sequence assembling result of verification three generations's sequencing, can
It is not too high region successfully to filter out three generations to assemble quality in result, and is marked out and.So in follow-up species
In research, there is the function of prompting in the region for using these of low quality if desired, and is provided quickly for follow-up improvement
Screening means.It also can prove that three generations assembles the accuracy and quality of result simultaneously, the accuracy of assembling result can be improved.
Brief description of the drawings
Fig. 1 is three generations's sequence assembling, and assembles the stream of result error correction to three generations respectively using three generation sequences and three generation sequences
Cheng Tu.
Fig. 2 is the figure that checking is compared with Fosmid sequences and assembling result；
Fig. 3 is the figure of the two generation sequence overburden depths distribution of assembling result.
Fig. 4 is the figure of the G/C content two generations overburden depth distribution of assembling result.
Fig. 5 goes to assess and verifies three generations for the present invention using three generations's sequential covering depth assembles the low covering of two generations in result deeply
Spend the flow chart in region.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Fig. 5 shows that the present invention goes to assess and verifies three generations using three generations's sequential covering depth and assembles two generations low covering in result
The flow chart of one embodiment of depth areas.
It is compared as shown in figure 5, step 202 is two generation sequences with three generations's assembling result, two generation sequences compare and arrive three generations
Assembling result simultaneously counts the overburden depth that three generations assembles each base of result.The average amount in the generation of general recommendations two can reach base
Because of more than the 50X of group, minimum 30X.The comparison in this stage can use the softwares such as comparison software bwa or SOAPAligner complete
Into.The base mismatch number of permission, which is generally less than, is equal to 2.And count three generations and assemble each site overburden depth of result.Statistics is can
To be carried out using SOAPCoverage softwares.
Step 204, the low extension of overburden depth region and selection：Using 1kb as window, low (the low depth of average overburden depth is selected
The threshold values of degree refers to the suggestion of table 1) region, and forwards, backwards respectively extension 30kb, obtain extension after sequence, due to three article used in lieu of a preface
The length range of row is hundreds of bp to tens kb, and these low overlay areas may as little as tens bp, so we are to low depth
30kb is respectively extended before and after overlay area, to ensure that three generation sequences can be compared with these regions.If Scaffold is front and rear
Less than 30k, the part taken, for example window is 5-6k regions, only takes 0-5k forward, removes 6-36k, i.e. 1-36kb backward；
It is insufficient behind Scaffold, get last base from window end.
The low depth threshold values recommendation tables of table 1
| Depth was sequenced in two generations | Low depth defines threshold values |
| 30X | 3X |
| More than 30X and it is less than or equal to 50X | 4-5X |
| More than 50X and it is less than or equal to 70X | 6-8X |
| More than 70X | 9-10X |
Step 206, three generation sequences compare with extension sequence：Compared with the sequence after three generation sequences and extension, here three
Generation sequence can be non-error correction sequence or from the sequence after error correction.Bwa can be used by comparing software.The data of three generation sequences
Amount suggests more than the 50X in Genome Size.
Step 208, base overburden depth counts：The three generations's average base for obtaining low overburden depth region of the former generations of 1kb bis- covers
Lid depth.
Step 210, result queue, low to two generation overburden depths or uncovered region, the average overburden depth of three generations are assembled
Mark less than or equal to 5X is low region, and the average overburden depth of three generations is more than 5X labeled as the high area of assembling quality
Domain.If continuous 2 or multiple all inadequate 5X of 1kb low depths region three generations, would combine them into a region.It is if continuous
2 or multiple while " the of a relatively high region of assembling quality " is marked as, then they is merged into note into " assembling quality a phase
To higher region ".Being marked as " the high region of assembling quality ", proportion is higher in window area selected by whole, then institute
The overall package quality for stating the sequence assembling result of three generations's sequencing is higher.
The Maize genome concrete application example of embodiment 1, the inventive method
(1) three generations's sequence assembling
Using three generations Maize genome data of three generations's composite software FALCON to 80X, (Pacbio unimolecules are sequenced in real time
(SMRT) result) assembled, and Polish error correction done to assembling result with three generations's initial data, then with the codes or datas pair of 60X bis-
The further error correction of assembling result after Polish, obtain the final assembling result of Maize genome.
(2) two generation sequences assemble result with three generations and are compared
Using SOAPAligner softwares by 60X PE250 two generation sequences (two generation sequences after filtering, gone joint and
Sequence after low quality base.Filtered by initial data to valid data through three step process：1) transition joint：Read matchings are sequenced
50% or the above of upper adapter sequences then delete whole piece reads；2) low quality data is filtered：If matter in read is sequenced
Base of the value less than 20 accounts for the 20% of whole piece read or then deletes whole piece read above；3) N is removed：If N in reads is sequenced
Content account for whole piece read 2% or more than, then delete whole piece read) compare to three generations and assemble result, each read allows most
Big 2bp mispairing, overburden depth of each site of result in two generation sequences is assembled with SOAPCoverage software statistics.
(3) the low extension of overburden depth region and selection
Using 1kb as window, select three generations and assemble each 1kb window areas average overburden depth in two generation sequences in result
Region less than or equal to 6X, and respectively extend 30kb forwards, backwards.As shown in table 2, such region always has 88.Before Scaffold
Afterwards if less than 30k, the part taken, for example window is 5-6k regions, only takes 0-5k forward, removes 6-36k, i.e. 1- backward
36kb；It is insufficient behind Scaffold, get last base from window end.
(4) three generation sequences compare with extension sequence
80X is compared by from three generation sequences of error correction and the sequence after extension with bwa softwares.
(5) base overburden depth counts
Obtain three generations's average base overburden depth in low overburden depth region of the former generations of 1kb bis-.
(6) result queue is assembled
As shown in table 2, mark of the average overburden depth of three generations less than or equal to 5X is relatively low region, altogether 35
It is individual；The average overburden depth of three generations is more than 5X labeled as the high region of assembling quality, 53 altogether.It can be marked well by the present invention
Remember that three generations assembles in result the low region of two generation overburden depths which is high assembling quality region, which is poor region.
The overburden depth situation of three generations in the former generation sequence low depth regions of 1kb bis- of table 2
| Less than 6X region quantities | Region of three generations's covering more than 5X | Region of three generations's covering less than or equal to 5X |
| 88 | 53 | 35 |
In summary, by the present invention can filter out three generations assemble result in quality be not too high region, and by its
Mark out and.In follow-up species research, there is the function of prompting in the region for using these of low quality if desired, and is
Follow-up improvement provides quick screening means.It also can prove that three generations assembles the accuracy and quality of result simultaneously, group can be improved
Fill the accuracy of result.
Claims (10)
1. a kind of method for the sequence assembling result for assessing three generations's sequencing, comprises the following steps：
(1) the sequence assembling result that two generation sequencing sequences of same sample are sequenced with three generations is contrasted；
(2) according to the comparison result of step (1), pick out from the sequence assembling result of three generations sequencing and surveyed in two generation
The low region of average overburden depth in sequence sequence, then by each selected region in the sequence assembling of three generations sequencing
As a result extended in, so as to obtain sequence after several extensions；
(3) sequence is individually compared after each extension for obtaining three generations's sequencing sequence with step (2)；
(4) according to the comparison result of step (3), each selected region is in three generations's sequencing sequence in statistic procedure (2)
In average overburden depth；
(5) according to the statistical result of step (4), the height of the assembling quality in each region selected in step (2) is determined, is entered
And realize the assessment of the sequence assembling result to three generations sequencing.
2. according to the method for claim 1, it is characterised in that：Methods described comprises the following steps：
(1) the sequence assembling result that two generation sequencing sequences of same sample are sequenced with three generations is contrasted, counts the three generations
Each overburden depth of the base in the two generations sequencing sequence in the sequence assembling result of sequencing, and then using 1-5kb as window,
Each window area being averaged in the two generations sequencing sequence in the sequence assembling result of three generations's sequencing is calculated to cover
Lid depth；
(2) according to the result of step (1), picked out from the sequence assembling result of three generations sequencing and sequence is sequenced in two generation
The low whole window areas of average overburden depth in row, then by each selected window area in three generations sequencing
Respectively extend 10-40kb in sequence assembling result forwards, backwards, so as to obtain sequence after several extensions；
(3) sequence is individually compared after each extension for obtaining three generations's sequencing sequence with step (2)；
(4) according to the comparison result of step (3), each selected window area is sequenced in the three generations in statistic procedure (2)
Average overburden depth in sequence；
(5) according to the statistical result of step (4), according to being assembled as follows to each window area selected in step (2)
The mark of quality height, and then assess the overall package quality of the sequence assembling result of three generations's sequencing：If in step (2)
Average overburden depths of selected some window area A in three generations's sequencing sequence is less than or equal to 5X, then by the window
Mouth region domain A is labeled as " the relatively low region of assembling quality "；If selected some window area B is in institute in step (2)
The average overburden depth stated in three generations's sequencing sequence is more than 5X, then by the window area B labeled as " assembling quality is of a relatively high
Region ".
3. according to the method for claim 2, it is characterised in that：It is according to assessing three generations sequencing as follows in step (5)
Sequence assembling result overall package quality：The quantity in " the of a relatively high region of assembling quality " that is marked and institute
It is bigger to state the ratio of both " the relatively low region of assembling quality " and " the of a relatively high region of assembling quality " total quantity,
Then the overall package quality of the sequence assembling result of three generations's sequencing is higher；
If continuous 2 or multiple while being marked as that " assembling quality is relatively low in selected window area in step (2)
Region ", then they are merged into note into one " the relatively low region of assembling quality "；If continuous 2 or multiple while marked
" the of a relatively high region of assembling quality " is designated as, then they are merged into note into one " the of a relatively high region of assembling quality ".
4. according to any described method in claim 1-3, it is characterised in that：In step (1), the two generations sequencing sequence is
Sequence of the initial data after filtration treatment obtained by two generation high-flux sequences；And/or
In step (3), three generations's sequencing sequence is non-error correction sequence or from error correction sequence.
5. according to any described method in claim 1-4, it is characterised in that：In step (2), the average overburden depth is low
Refer to that average overburden depth is less than " low depth defines threshold values ", be following any：
(a1) when the average overburden depth of two generation sequencing is 30X, described " low depth defines threshold values " is 3X；
(a2) when the average overburden depth of two generation sequencing is more than 30X and is less than or equal to 50X, described " low depth defines valve
Value " is 4-5X；
(a3) when the average overburden depth of two generation sequencing is more than 50X and is less than or equal to 70X, described " low depth defines valve
Value " is 6-8X；
(a4) when the average overburden depth of two generation sequencing is more than 70X, described " low depth defines threshold values " is 9-10X.
6. according to any described method in claim 1-5, it is characterised in that：In step (1), the two generations sequencing is averaged
Data volume reaches more than the 30X of Genome Size；
Specifically, the average amount of the two generations sequencing reaches more than the 50X of Genome Size.
7. according to any described method in claim 1-6, it is characterised in that：In step (1), during the comparison allow
Base mismatch number be less than or equal to 2.
8. according to any described method in claim 1-7, it is characterised in that：The average amount of three generations's sequencing is in base
Because of group a more than 50X for size.
9. the assessment system of the sequence assembling result of three generations's sequencing, including at data processing equipment A, data processing equipment B, data
Manage device C, data processing equipment D and data processing equipment E；
Module a1, module a2, module a3 are set in the data processing equipment A；The module a1 can to two generation sequencing sequences with
The sequence assembling result of three generations's sequencing is contrasted；The result that the module a2 can compare according to the module a1, counts institute
State each overburden depth of the base in the two generations sequencing sequence in the sequence assembling result of three generations's sequencing；The module a3 energy
Enough statistical results according to the module a2, using 1-5kb as window, it is calculated in the sequence assembling result of three generations's sequencing
Each average overburden depth of the window area in the two generations sequencing sequence；
Module b1 and module b2 are set in the data processing equipment B；The module b1 can obtain according to the data processing equipment A
The result obtained, the average overburden depth in the two generations sequencing sequence is picked out from the sequence assembling result of three generations sequencing
Low whole window areas；The module b2 can survey each window area selected by the module b1 in the three generations
Respectively extend 10-40kb in the sequence assembling result of sequence forwards, backwards, so as to obtain sequence after several extensions；
Module c1 is set in the data processing equipment C；The module c1 can fill three generations's sequencing sequence and the data processing
Sequence is individually compared after putting each extension of B acquisitions；
Module d1 is set in the data processing equipment D；The comparison that the module d1 can obtain according to the data processing equipment C
As a result, each window area described in the data processing equipment B selected by module b1 is counted in three generations's sequencing sequence
In average overburden depth；
Module e1 and module e2 are set in the data processing equipment E；The module e1 can obtain according to the data processing equipment D
The statistical result obtained, according to as follows to each window area progress selected by module b1 described in the data processing equipment B
The mark of assembling quality height：If average overburden depths of some window area A in three generations's sequencing sequence is less than
Equal to 5X, then the window area A is labeled as " the relatively low region of assembling quality "；If some window area B exists
Average overburden depth in three generations's sequencing sequence is more than 5X, then by the window area B labeled as " assembling quality is relatively
High region "；The module e2 can be counted according to the mark result of the module e1 and be calculated institute in the data processing equipment B
State the quantity in " the of a relatively high region of assembling quality " that module b1 is marked and " the relatively low area of assembling quality
The ratio of both domain " and " the of a relatively high region of assembling quality " total quantity；Wherein, window area selected by the module b1
In if continuous 2 or multiple while being marked as " the relatively low region of assembling quality ", then they are merged into note into one
" the relatively low region of assembling quality "；If continuous 2 or multiple while being marked as " the of a relatively high area of assembling quality
Domain ", then they are merged into note into one " the of a relatively high region of assembling quality ".
10. system in claim 1-8 described in any described method or claim 9 it is following it is any in application：
(A) the sequence assembling result of three generations's sequencing is assessed；
(B) screen and mark ropy region in the sequence assembling result that three generations is sequenced.
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