CN109517882A - A kind of quality control method and application for detecting unique both-end library tag combination - Google Patents

A kind of quality control method and application for detecting unique both-end library tag combination Download PDF

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CN109517882A
CN109517882A CN201811337895.2A CN201811337895A CN109517882A CN 109517882 A CN109517882 A CN 109517882A CN 201811337895 A CN201811337895 A CN 201811337895A CN 109517882 A CN109517882 A CN 109517882A
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张之宏
罗健
汉雨生
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Guangzhou Burning Rock Dx Laboratory Co ltd
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Abstract

The invention discloses provide a kind of quality control method for detecting unique both-end library tag combination and application, belong to technical field of biological, the quality control method is the following steps are included: S1) gDNA library of the building with unique both-end library tag combination, the library built is subjected to upper machine sequencing, and read library sequence label, S2 first time Analysis of quality control) is carried out to library sequence label, S3) according to the analysis result of S2, in case there is a need, replace problematic label stock, according to step S1) method, rebuild the library gDNA with unique both-end library tag combination, the library built is subjected to upper machine sequencing, and read library sequence label, S4 second of Analysis of quality control) is carried out to library sequence label, according to result judge whether continue according to S3) method replacement have Problem library label, until all library labels meet the index of Analysis of quality control.Quality control method of the present invention can improve the detection efficiency of library label, be more suitable for the demand that library is accurately sequenced.

Description

A kind of quality control method and application for detecting unique both-end library tag combination
Technical field
The invention belongs to technical field of biological more particularly to a kind of for detecting unique both-end library tag combination Quality control method and application.
Background technique
With the rapid development of high-throughput techniques, the flux of sequenator is increasing, early to separate method example first with physics Method as shunting (Lane) formula flowing groove (Flow Cell) distinguishes different sequencing libraries has been not suitable for.Multiple library sequencing (Multiplex Sequencing) is widely used in the every field of two generations sequencing.The key of multiple library sequencing is then text Library label (Index).Library label is in the preparation of the library (Next Generation Sequecing) NGS, to each sample Special sequence label is carried out, for the distinguished sequence of distinguishing different DNA, general length is 4~12 bases longs.In high pass It measures in program process, sequencing reaction, the Insert Fragment in library is carried out by the library of different known label sequence marks after mixing And label is read out sequentially and is converted to base.In next analytic process, software utilizes expected sequence label pair Sequencing result is classified, and sequencing result is split into different samples.
In multiple sequencing procedure, in case of the distribution of library sequence mistake, it is not belonging to the sequence in certain library originally just It can be by the classification of mistake.The generation of this kind of mistake distribution will bring certain applications the analysis result of mistake.For example, when The library for being derived from the tissue samples of cancer patient and the library for the tissue samples for being derived from benign tumour patient are sequenced jointly, such as Fruit has the sequence of part cancerous tissue sample to be assigned in benign tumor tissue sample by mistake, leads to the detection report of benign tumour patient Announcement is shown as malignant tumour, leads to diagnostic error.
There is a lot of reasons that library sequence can be caused to be distributed by mistake.Common includes following several: 1) prepared by library Cross contamination in journey, 2) cross contamination in Tag primer production process, 3) multiple library carries out cluster reaction in flowing groove The cross reaction of Shi Fasheng and 4) due to cluster density is excessive etc. caused by optical aberration etc..
Suitable for two generation sequencing library Tag primers, often length is 50~70 bases, it is however generally that needs to purify to guarantee The purity of overall length primer.However, purifying itself frequently can lead to more intersect dirty due to needing gel extraction or crossing column Dye.For HPLC (high performance liquid chromatography), purification column can inevitable band to the absorption and reuse of Tag primer Carry out cross contamination.Although this kind of pollution can be by carrying out empty sample elution or nothing between two different Tag primers cross column purification Sample elution is closed to reduce residual contamination, this still cannot be avoided cross contamination completely.Rule of thumb, it is residual to purify meeting twice for front and back Stay 0.5%~5% previous Tag primer into the latter Tag primer.
The hypersensitivity as brought by the high throughput of NGS, the quality inspection of Tag primer need very sensitive method with It is polluted in detection down to the one thousandth even possibility of a ten thousandth.It is closely similar additionally, due to the sequence between Tag primer, No matter conventional method such as qPCR is unsuitable for from sensitivity or specificity for detecting pollution.Generally conventional method It is still to carry out quality inspection using NGS platform, but conventional method can only at most detect every Lane one target labels and draw Object, in this way for so that quality inspection cost is become unattainable.
Therefore, it is necessary to a kind of quality control method of novel unique both-end library tag combination be designed, to propose detection effect Rate.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and provide a kind of for detecting unique both-end Index Combined quality control method and application, can improve the detection efficiency of library label, be more suitable for the demand that library is accurately sequenced.
To achieve the above object, the technical scheme adopted by the invention is as follows: one kind is for detecting unique both-end library set of tags The quality control method of conjunction comprising following steps:
S1) using library tag standards product and gDNA standard items as raw material, building is with unique both-end library tag combination The library built is carried out upper machine sequencing, and reads library sequence label by the library gDNA；
S2 first time Analysis of quality control) is carried out to library sequence label, the index of Analysis of quality control includes following items: maximum Unilateral label pollution accounting≤2.5%, maximum tag combination pollution accounting≤0.01%, every group of exemplar sequence item number >= 5000, all tag combinations mix accounting coefficient of variation≤0.5, Compositive sequence percent of pass >=97%, every group of exemplar sequence Column accounting >=0.2/ library tag combination logarithm, the label accounting that unilateral side is greater than 1% pollution answer≤10%；
S3) if step S2) Analysis of quality control show index do not meet, recombine do not meet Quality Control requirement library mark Label；According to step S1) method, with the satisfactory library label of library label, first time Analysis of quality control that recombines and GDNA is raw material, and the library built is re-started upper machine by gDNA library of the building with unique both-end library tag combination Sequencing, and read library sequence label；
S4 second of Analysis of quality control) is carried out to library sequence label, until all library labels meet the finger of Analysis of quality control Mark；
In the parameter of Analysis of quality control, the uniqueness both-end library tag combination is by upstream library label and downstream library Label composition, upstream library label are referred to as IG5, and IG5 is included as A and B；Downstream library label is referred to as IG7, IG7 includes a and b；Matching and correctly uniqueness both-end library tag combination are A-a and B-b；Unmatched uniqueness both-end library Tag combination is A-b and B-a；By analyzing the available respective sequence item number of combination of the above after each sequencing reaction；
The unilateral side label pollution accounting is the cross contamination ratio occurred between group interior label, and pollution is only possible to occur In group, i.e., polluted in IG5 group or/and in IG7 group；
When any cross contamination does not occur in process of production for a of IG7, for the A of IG5, wherein the pollution containing B accounts for Than=sequence item the number containing B-a/all sequence item numbers containing a,
When any cross contamination does not occur in process of production for the A of IG5, for a of IG7, wherein the pollution containing b accounts for Than=contain A-b sequence item number/all sequence item numbers containing A；
A is polluted when B pollutes A and b, then B-b tag combination pollution accounting=(the sequence item number containing B-a/all contains a Sequence item number) × (containing A-b sequence item number/all sequence item numbers containing A)；
When any cross contamination does not occur in process of production for the b of IG7, for the B of IG5, wherein the pollution containing A accounts for Than=sequence item the number containing A-b/all sequence item numbers containing b,
When any cross contamination does not occur in process of production for the B of IG5, for the b of IG7, wherein the pollution containing a accounts for Than=contain B-a sequence item number/all sequence item numbers containing B；
B is polluted when A pollutes B and a, then A-a tag combination pollution accounting=(the sequence item number containing A-b/all contains b Sequence item number) × (containing B-a sequence item number/all sequence item numbers containing B)；
Every group of exemplar sequence item number is to be contained by filtered every group correct matched sequence item number of system The sequence item number of A-a or sequence item number containing B-b；
All tag combination mixing accounting coefficient of variation are to pass through filtered every group correct matched sequence item of system Number variance of proportion coefficient in total correct sequence item number of pairing filtered by system；
The Compositive sequence percent of pass be sequencing reaction after by system it is filtered it is correct pairing and ordered sequence it is total Item number is accounted for filtered by system after all sequences total number ratio；
It is logical that every group of exemplar sequence accounting is that the sequence item number correctly matched by filtered every group of system accounts for Cross the ratio of total sequence after system filters；
The unilateral label accounting for being greater than 1% pollution are as follows: in the label of upstream library, pollution ratio is greater than 1% library The ratio of the total library number of tags of number of tags Zhan；And in the label of downstream library, it is total that pollution ratio is greater than 1% library number of tags Zhan The ratio of library number of tags.
As an improvement of the above technical solution, the step S1) successively the following steps are included: gDNA standard items prepare, GDNA fragmentation, end are repaired, connector connection, the purifying of connector connection product, amplified library, expand the purifying in library, purifying text The quality inspection in library, machine sequencing in the detection of purified library clip size and library.
As an improvement of the above technical solution, unique both-end library tag combination is made of IG5 group and IG7 group, IG5 Sequence Hamming distance >=2 of library label between the Hamming distance >=3, IG5 and IG7 group of the library label in IG7 respectively group.
As a further improvement of the above technical scheme, library label is purified and is led to by high performance liquid chromatography Cross mass spectral analysis confirmation molecular weight, it is desirable that purity >=85%.
As an improvement of the above technical solution, unique both-end library tag combination is made of 96 pairs of library labels, i.e., There is 96 upstreams library label in IG5 group, there is 96 downstreams library label in IG7 group, corresponds；Every group of exemplar sequence Accounting is then accordingly adjusted to >=0.2%.
As an improvement of the above technical solution, unique both-end library tag combination is made of 48 pairs of library labels, i.e., There is 48 upstreams library label in IG5 group, there is 48 downstreams library label in IG7 group, corresponds；Every group of exemplar sequence Accounting is then accordingly adjusted to >=0.4%.
As an improvement of the above technical solution, when unique both-end library tag combination is made of 192 pairs of library labels, i.e., There is 192 upstreams library label in IG5 group, there are 192 downstream library labels in IG7 group, corresponds, every group of exemplar sequence Accounting is then accordingly adjusted to >=0.1%.
As an improvement of the above technical solution, when unique both-end library tag combination is made of 288 pairs of library labels, i.e., There is 288 upstreams library label in IG5 group, there are 288 downstream library labels in IG7 group, corresponds, every group of exemplar sequence Accounting is then accordingly adjusted to >=0.07%.
As an improvement of the above technical solution, when unique both-end library tag combination is made of 384 pairs of library labels, i.e., There is 384 upstreams library label in IG5 group, there are 384 downstream library labels in IG7 group, corresponds, every group of exemplar sequence Accounting is then accordingly adjusted to >=0.05%.
It is applied in sample sequence measurement in addition, the present invention also provides the quality control methods.
The beneficial effects of the present invention are: the present invention provides a kind of for detecting the Quality Control of unique both-end library tag combination Method and application, the quality control method energy efficient detection go out the cross contamination of library label, and advantage of lower cost, can more be suitble to sample The high throughput assay of this sequence.
Detailed description of the invention
Fig. 1 shows the Quality Control result simulated for the first time in embodiment 1；
Fig. 2 shows the Quality Control result of second of simulation in embodiment 1；
Fig. 3 shows the result of the end the IG5 first time Analysis of quality control of embodiment 2；
Fig. 4 is the pollution accounting hotspot graph of the end the IG7 first time Analysis of quality control of embodiment 2, has 96 pairs of labels to draw in Fig. 4 Object, abscissa are followed successively by IG5A01~IG5A12, IG5B01~IG5B12, IG5C01~IG5C12 until IG5H01 from left to right ~IG5H12, ordinate be followed successively by from top to bottom IG7A01~IG7A12, IG7B01~IG7B12, IG7C01~IG7C12 until IG7H01~IG7H12；The oval point irised out is expressed as undesirable Tag primer in figure；It is similar below；
Fig. 5 is the pollution accounting hotspot graph of the end the IG5 first time Analysis of quality control of embodiment 2；
Fig. 6 is the distribution map of the pollution accounting of the end the IG7 and IG5 first time Analysis of quality control of embodiment 2；
Fig. 7 shows the stability contrast result of the Analysis of quality control twice at the end IG7 in embodiment 2；
Fig. 8 shows the stability contrast result of the Analysis of quality control twice at the end IG5 in embodiment 2；
Fig. 9 shows the result of the end the IG5 first time Analysis of quality control of embodiment 3；
Figure 10 is the pollution accounting hotspot graph of the end the IG7 first time Analysis of quality control of embodiment 3；
Figure 11 is the pollution accounting hotspot graph of the end the IG5 first time Analysis of quality control of embodiment 3；
Figure 12 is the distribution map of the pollution accounting of the end the IG7 and IG5 first time Analysis of quality control of embodiment 3.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiments and the drawings pair The present invention is described further.
In addition, it is necessary to which explanation is in this Shen description of the invention, Index, library label and Tag primer indicate same A meaning；In the calculating of every group of exemplar sequence accounting, the result of 0.2/ library tag combination logarithm retains a non-zero Number (and rounding up).
Unique both-end library label leads to the principle of sample contamination in anti-cross-contamination
In the field NGS, in order to distinguish the different samples under the same sequencing reaction, add during building library to different samples Upper specific " label " (Index), to be separated different sample datas in subsequent data analysis.As sequenator is logical The continuous improvement of amount, more samples are pieced together into the same flow channel (Lane) sequencing, to the quantity and differentiation of Index Degree is put forward higher requirements.In addition, Illumina HiSeqX/4000 and NovaSeq are used different from other Illumina The clustering method of sequenator, document report its have higher Index cross contamination risk.The single-ended Index primer foundation of tradition One end carries out data fractionation, is easy to when polluting by data mistake point.It can be utmostly using unique both-end Index primer It avoids guaranteeing the reliability of product due to Index cross contamination bring sample contamination risk.Unique both-end Index draws Object increases " dual fail-safe " for sequencing sequence, pollutes due to carrying out data fractionation by unique both-end pairing Index Most of sequence can be dropped.Table 1 then compare it is single-ended, combine both-end and unique both-end Index strategy Index is intersected it is dirty The tolerance of dye.
Table 1
High-throughput pollution quality inspection principle is carried out to unique both-end Index primer by NGS method
Due to having used unique both-end Index, each sample is by Index label 2 times, so to single-ended label Cross contamination tolerance rises significantly between primer.For example, practical if the ratio of 2 pairs of unilateral side Index pollution is 1% Caused sample mistake divides pollution risk to be 1% × 1%=0.01%.This tolerance also significantly reduces the synthesis of Index primer With the pressure of purifying, control manufacturing cost further.
Using the advantage of unique both-end Index, Tag primer is detected using NGS the present invention provides a kind of simple possible The quality control method of cross contamination.Its basic principle is combined in entire sequencing result based on unexpected both-end Index is observed Accounting is to estimate the maximum cross contamination possibility that may occur and the Index being related to, to avoid due to Index Mistake distribution between sample caused by cross contamination between primer.
For example, four libraries are respectively labeled as A+a, B+b, C+c, D+d.Therefore when carrying out sequence analysis, only Having above-mentioned 4 kinds of combinations is considered as valid combination.For combining A+b, because theoretically only having A that can match with a, if observation Having arrived A+b combination, there are two types of possibilities: 1) Tag primer b enters primer a, and defining S here is the sequence containing this kind of Index Number, estimation pollution ratio are S(A+b)/SA；2) primer A enters primer B, and estimation pollution ratio is S(A+b)/Sb.It may be noted that It is that the premise of the calculation method is in generic Index such as A/B/C/D without containing any non-generic Index for example a/b/c/d.In addition appraising model only considered simple one-to-one pollution mode, rather than the complex situations such as multiple pollution.Separately The outer calculation method is the directionality estimated maximum possibility of pollution and had no ability to judgement pollution, in fact any After a kind of unidirectional contamination accident occurs, such as the event of " A enters B ", it can all be detected as " A enters B " or " b enters a " Two kinds of possibilities.According to the computation model, we are estimated that unique combination both-end Index library A+a in multiple sequencing The greatest combined pollution risk by other primers are as follows:
However due to it is desirable that combination there was only A+a, B+b, C+c, tetra- kinds of D+d, actual effective greatest contamination wind Danger may be calculated:
In practical application example, we carry out PCR operation label Index to 48 pairs or 96 pairs of Index primers respectively and arrive Then library mixes and carries out routine MiSeq sequencing.Sequencing post analysis calls directly analysis script to 96 × 6=9216 Kind combined sequence is analyzed, and is found improper combined ratio and is calculated respective pollution accounting.
The upper machine sequencing of Index
1, prepared by gDNA standard items
1) 48plex Index progress quality inspection needs 500ng gDNA standard items, and 96plex Index carries out quality inspection needs 1000ng gDNA standard items；
2) 50 μ l 1 × IDTE Buffer are taken, are added in new 1.5ml Eppendorf LoBind pipe, then Xiang Guanzhong The gDNA standard items of corresponding volume are added: the detection of 48plex Index plate, it is 2 μ l that volume, which is added, in gDNA standard items；96plex The detection of Index plate, it is 4 μ l that volume, which is added, in gDNA standard items；It is vortexed afterwards and mixes 10~15s, rear of short duration centrifugation makes solution return to pipe Bottom；
3) standard items dilution product are transferred in Covaris MicroT Μ BE pipe, supplement 1 × IDTE Buffer to 50 μ L carries out subsequent DNA fragmentationization operation afterwards.
2, gDNA fragmentation
DNA is interrupted into the segment to 170~200bp using Covaris M220 instrument, after the completion of interrupting, by Covaris MicroTube pipe takes out, and centrifugation makes liquid return to tube bottom.
3, end is repaired, 3 ' ends plus A
1) reagent prepares: opening KAPA Hyper Prep 96reaction Kit, takes out following 2 pipes and be placed in and melt on ice Change；
2) in new 1.5ml Eppendorf LoBind pipe, end is prepared on ice and repairs and A reaction system is added to mix Liquid, finger flick 3~5 times, turn upside down mixing 2~3 times, and centrifuge is centrifuged 1~3s；The configuration of reaction system is as shown in table 2；
3) 60 μ l mixing liquids of absorption are distributed into 4 (48plex Index plates) or 8 (96plex Index plate) 0.2ml are flat In lid PCR pipe, 1~3s of the of short duration centrifugation of centrifuge；
4) be put into PCR instrument, perform the following operation: 85 DEG C of heat lids, 20 DEG C of 30min, 65 DEG C of 30min, 4 DEG C save, in 2h Into in next step.
Table 2
4, connector connects, and the DNA double chain segment both ends for adding A are connect with preparation joint (containing T cohesive end)
1) in new 1.5ml Eppendorf LoBind pipe, connector coupled reaction system mixing liquid, finger are prepared on ice It flicks 3~5 times, turns upside down mixing 2~3 times, centrifuge is centrifuged 1~3 second；The configuration of reaction system such as table 3 shows；
2) (48plex Index plate 4 is managed totally, 96plex Index plate in the above-mentioned 0.2ml pipe of 50 μ l mixing liquids of absorption addition Totally 8 pipe), pipettor pipettor blows and beats 5 mixings up and down, is centrifuged 1~3s；
3) following procedure: 20 DEG C of 15min, 70 DEG C of 10min is run in PCR instrument, 4 DEG C save (85 DEG C of heat lids).
Table 3
5, the purifying of connection product removes the other compositions such as connector dimer and not connected connector
1) it turns upside down 2~3 times, is vortexed and mixes the SPB magnetic bead that 5~10s replys room temperature, make its homogenization；Take 1.5ml from The magnetic bead and adjunction head product of homogenization is successively added in coupled reaction system and magnetic bead volume 1:0.8 ratio in heart pipe；Specifically Strategy is as follows: magnetic bead is 352 μ l, connector product is 440 μ l, and 4 pipes merge into the purifying of 1 pipe, totally 1 pipe；Magnetic bead is 2 × 352 μ l, connects Head product is 2 × 440 μ l, and 4 pipes merge into the purifying of 1 pipe, totally 2 pipe (96plex Index)；It is vortexed and mixes after addition, rotation is incubated for 5min, of short duration centrifugation；
2) centrifuge tube is placed in magnetic frame, waits solution clarification；Centrifuge tube is placed in motionless on magnetic frame, opening pipe lid, Clarified supernatant carefully is siphoned away, avoids encountering magnetic bead；
3) pipe is still placed on magnetic frame, and 75% ethyl alcohol of 500 μ L Fresh is added in every pipe, and 1min is waited to keep magnetic bead abundant Precipitating, during which slow rotating centrifugal pipe 1 encloses in the horizontal direction, siphons away ethyl alcohol；Multiple this step 1 time；
4) it is centrifuged 1~3s, centrifuge tube is placed back in into magnetic frame and stands 30s, using the cleared residual ethanol of pipettor, is kept Pipe Gai Kaiqi；Room temperature 3min keeps magnetic bead dry, and 500 μ l EB solution are added in every pipe, and sufficiently piping and druming mixes, and is incubated at room temperature 2min； Centrifuge tube is placed in magnetic frame 2min until solution clarification, pipettes 490 μ l supernatants using pipettor, be transferred to new It is standby on ice in Eppendorf LoBind 1.5ml centrifuge tube (96plex Index plate, two pipes merge into 1 pipe after elution) With.
6, amplified library, amplification have connected the library of connector
1) prepare respective volume reaction system in 5ml Eppendorf LoBind pipe (or 15ml centrifuge tube) to mix Liquid (is prepared) on ice, and finger flicks 3~5 times, is turned upside down mixing 2~3 times, stands 0.5~1min vertically；Reaction system is matched It sets as shown in table 4；
2) prepared reaction system mixed liquor is evenly distributed in 8 connecting legs, partial volume is 138 μ l every time (96Index pair Plate (refer part2#) detection needs to carry out mean allocation twice: 142 μ l+132 μ l)；
3) reaction system mixed liquor is distributed into 48 new orifice plates (48plex Index) or 96PCR plate (96plex Index), packing volume is 22.5 holes μ l/；
4) taken out from IDP plate 2.5 μ l Index (be added to good 48 orifice plate of reaction system mixed liquor of above-mentioned packing or In 96 hole PCR plates, piping and druming is mixed 2~3 times repeatedly, and sealer；Knockout plate machine is centrifuged 1000rpm, 1min (25 μ l of reaction volume)；It sets In running in PCR instrument, operation program is as shown in table 5.
Table 4
Table 5
7, the library purifying expanded, removes primer dimer and reaction system
1) SPB magnetic bead is turned upside down 2~3 times, 5~10s is mixed under VORTEX maximum (top) speed, is made its homogenization；
2) corresponding SPB magnetic bead is drawn into loading slot, 20 μ l SPB magnetic beads of each sample addition (sample: magnetic bead=1: 0.8): 1440 μ l or so magnetic bead is then added in 48 samples in loading slot, and it is left that 2880 μ L are then added in 96 samples in loading slot Right magnetic bead；
3) 48 orifice plates are taken out from PCR instrument, 1000rpm 3s carefully tears pad pasting off；20 μ l SPB are drawn from loading slot Magnetic bead is added in 48 orifice plates/96 hole PCR plates, up and down piping and druming 10 times；
4) 48 orifice plates/96 hole PCR plate pad pastings, of short duration centrifugation 1000rpm 3s are placed in room temperature 5min；48 orifice plates/96 hole PCR Plate is placed on 96 hole magnetic frames, is clarified to solution；Film is abandoned, 45 μ l of supernatant is drawn, is abandoned；
5) 48 orifice plates/96 hole PCR plates are still placed on magnetic frame, and 75% second of 200 μ l Fresh is added in sample aperture Alcohol；48 orifice plates/96 hole PCR plates are stood on magnetic frame embathes magnetic bead sufficiently, to 1min, abandons ethyl alcohol；Repeat this step 1 time；
6) 48 orifice plates/96 hole PCR plates are rested on into 30s on magnetic frame, and cleared residual ethanol；By 48 orifice plates/96 hole PCR Plate is removed from magnetic frame, is placed in room temperature 2min on PCR plate frame, keeps magnetic bead dry；14 μ are added in 48 orifice plates/96 hole PCR plates L EB covers eight connecting leg lids, vortex 5s or so, of short duration centrifugation 1000rpm 3s；
7) 48 orifice plates are placed in incubation at room temperature 2min, abandon film, 48 orifice plates is placed in magnetic frame 2min, until solution is clarified； 8 μ L of supernatant is pipetted into 48 new orifice plates/96 hole PCR plates, not magnetically attractive pearl；
8) each column library is transferred in same new 8 connecting leg of 0.2ml, then 8 connecting leg Chinese library of 0.2ml is transferred to Same new 1.5ml Eppendorf LoBind pipe, merges into the library pooling, Vortex is mixed and is centrifuged；After mixing Purified library take out 20 μ l to one it is new 1.5ml Eppendorf LoBind pipe, add 180 μ l EB, repeatedly blow and beat 5~ 6 times, 10 times of library beforehand dilution are prepared for subsequent detection.8, the quality inspection of purified library
It usesDsDNA HS (High Sensitivity) Assay Kit (Thermo Fisher) measurement dilution Library concentration afterwards, and the pre- library concentration that converts back；Library concentration circle is between 9~60ng/ μ l, and Labchip result is normal, Then library construction part is qualified, can carry out machine on subsequent Miseq；It needs to re-start library system if it cannot reach requirement It is standby.
9, the library fragments size detection (Library QC) purified
Using The LabChip DNA High Sensitivity Reagent kit (Perkin Elmer) to dilution It is detected in library afterwards；Qualified library fragments main peak is in 350~500bp, without obvious small fragment in the section 10~150bp.
10, machine strategy (Miseq Run) on library
1) purified library is diluted to 4nM according to the detectable concentration of QC, 1N NaOH is diluted using nuclease-free water To 0.2N；
2) library is denaturalized: taking 5 μ l of the library for being diluted to 4nM that new 1.5ml Eppendorf LoBind pipe is added, then 5 μ l 0.2N NaOH are added, piping and druming mixes 15~20 times, is incubated at room temperature 5min；
3) library is diluted to 13pM；
4) subsequent operation refers to Illumina Miseq operating guidance, is recycled using Read1=12, Index1=8 circulation, Library is sequenced in the corresponding setting of Index2=8 circulation.
11, sequencing data analysis (QC Analysis)
The sequence of all index1 and index2 are exported using Illumina bcl2fastq software cooperation relevant parameter (Fastq format), it is for statistical analysis to sequence using corresponding scripts, obtain each index.
11, library sequencing result criterion
Machine index under Miseq: sequencing data quality 01:Q30 > 90%, sequencing data quality 02:PF > 97%, sequencing data Quality 03:Phasing and Prephasing are respectively less than 0.30.
Embodiment 1The simulation of quality control method
1) unidirectional pollution is simulated for the first time: detecting 1 cross contamination for the first time, provides 2 kinds of supposition pollution directions, it is maximum Contamination ratio (i.e. maximum unilateral label pollution accounting) 4%；Analogue data generates 96 pairs of standard matched sequences, occurs polluting Normal pairing IG7F01+IG5F01 48000, IG7F01+IG5E012000 item；Remaining each pair of normal pairing is 50000 Item.The literature data of simulation is subjected to data analysis, the results are shown in Table 6 for actual test, carries out Quality Control point according to the parameter of table 6 Analysis, it is as shown in Figure 1 to obtain Analysis of quality control result；Wherein, (i.e. maximum tag combination pollution accounts for maximum pairing pollution accounting product Than)=4% × 0=0, it is correctly 48000 with sequence item number, and correctly pairing and ordered sequence item number are 48000, sequence passes through Rate is 100%, and the number of tags that unilateral side is greater than 1% pollution is a kind, and it is (i.e. unilateral to be greater than 1% pollution to be greater than 1% pollution index accounting Label accounting)=1/96=1.04%.
Table 6
2) second simulation can cause the two-way pollution of sample mistake point: detect 2 cross contaminations for the second time, and this 2 A cross contamination can cause sample mistake point, greatest contamination ratio 2%, maximum pairing pollution product 0.04%；Simulate number It is 50000 according to standard matched sequence is generated, occurs normal pairing IG7F01+IG5F01 48000 polluted, mistake is matched To IG7F01+IG5E01 1000, IG7E01+IG5F011000 item.The library sample of simulation is subjected to data analysis, it is practical Test result is as shown in table 7, carries out Analysis of quality control according to the parameter of table 7, it is as shown in Figure 2 to obtain Analysis of quality control result.
Table 7
It can be seen that this simulation test test result and expection are consistent.
Embodiment 2
The present embodiment carries out quality inspection with 96 pairs of library labels, and first time Analysis of quality control report result is as shown in table 8 and table 9, table 8 The case where only listing pollution with table 9.
Table 8 is directed to the sequencing result of the end IG7 Index
Query It is expected that compound object It is expected that combining Unexpected combination Unexpected compound object Total sequence item number Unexpected composite sequence item number Pollution sources It is contaminated Pollute accounting
IG7A01 IG5A01 IG7A01-IG5A01 IG7A01-IG5B01 IG5B01 96 45 IG5B01 IG5A01 46.88%
IG7A01 IG5A01 IG7A01-IG5A01 IG7A01-IG5A02 IG5A02 96 51 IG5A02 IG5A01 53.13%
IG7A08 IG5A08 IG7A08-IG5A08 IG7A08-IG5H07 IG5H07 53249 88 IG5H07 IG5A08 0.17%
IG7B02 IG5B02 IG7B02-IG5B02 IG7B02-IG5A03 IG5A03 40825 43 IG5A03 IG5B02 0.11%
IG7B10 IG5B10 IG7B10-IG5B10 IG7B10-IG5D08 IG5D08 46021 70 IG5D08 IG5B10 0.15%
IG7B11 IG5B11 IG7B11-IG5B11 IG7B11-IG5C11 IG5C11 47969 68 IG5C11 IG5B11 0.14%
IG7C01 IG5C01 IG7C01-IG5C01 IG7C01-IG5G12 IG5G12 39518 64 IG5G12 IG5C01 0.16%
IG7C06 IG5C06 IG7C06-IG5C06 IG7C06-IG5C07 IG5C07 60810 637 IG5C07 IG5C06 1.05%
IG7C08 IG5C08 IG7C08-IG5C08 IG7C08-IG5B08 IG5B08 67961 119 IG5B08 IG5C08 0.18%
IG7D03 IG5D03 IG7D03-IG5D03 IG7D03-IG5E03 IG5E03 44222 48 IG5E03 IG5D03 0.11%
IG7D03 IG5D03 IG7D03-IG5D03 IG7D03-IG5C03 IG5C03 44222 56 IG5C03 IG5D03 0.13%
IG7D04 IG5D04 IG7D04-IG5D04 IG7D04-IG5D03 IG5D03 40521 41 IG5D03 IG5D04 0.10%
IG7D07 IG5D07 IG7D07-IG5D07 IG7D07-IG5E08 IG5E08 39029 281 IG5E08 IG5D07 0.72%
IG7D08 IG5D08 IG7D08-IG5D08 IG7D08-IG5C08 IG5C08 53581 85 IG5C08 IG5D08 0.16%
IG7D09 IG5D09 IG7D09-IG5D09 IG7D09-IG5E09 IG5E09 54786 70 IG5E09 IG5D09 0.13%
IG7E03 IG5E03 IG7E03-IG5E03 IG7E03-IG5F03 IG5F03 60714 78 IG5F03 IG5E03 0.13%
IG7E07 IG5E07 IG7E07-IG5E07 IG7E07-IG5D07 IG5D07 57285 88 IG5D07 IG5E07 0.15%
IG7F04 IG5F04 IG7F04-IG5F04 IG7F04-IE5D04* IE5D04* 49814 54 IE5D04* IG5F04 0.11%
IG7F07 IG5F07 IG7F07-IG5F07 IG7F07-IG5E07 IG5E07 55273 63 IG5E07 IG5F07 0.11%
IG7G08 IG5G08 IG7G08-IG5G08 IG7G08-IG5F08 IG5F08 43769 167 IG5F08 IG5G08 0.38%
IG7G10 IG5G10 IG7G10-IG5G10 IG7G10-IG5F06 IG5F06 57227 60 IG5F06 IG5G10 0.10%
IG7H02 IG5H02 IG7H02-IG5H02 IG7H02-IG5H03 IG5H03 38360 58 IG5H03 IG5H02 0.15%
IG7H07 IG5H07 IG7H07-IG5H07 IG7H07-IG5G07 IG5G07 36388 42 IG5G07 IG5H07 0.12%
Table 9 is directed to the sequencing result of the end IG5 Index
Query It is expected that compound object It is expected that combining Unexpected combination Unexpected compound object Total sequence item number Unexpected composite sequence item number Pollution sources It is contaminated Pollute accounting
IG5A01 IG7A01 IG5A01-IG7A01 IG5A01-IG7B01 IG7B01 26 26 IG7B01 IG7A01 100.00%
IG5A02 IG7A02 IG5A02-IG7A02 IG5A02-IG7A01 IG7A01 49928 51 IG7A01 IG7A02 0.10%
IG5A03 IG7A03 IG5A03-IG7A03 IG5A03-IG7B02 IG7B02 33067 43 IG7B02 IG7A03 0.13%
IG5A08 IG7A08 IG5A08-IG7A08 IG5A08-IG7B08 IG7B08 53201 60 IG7B08 IG7A08 0.11%
IG5B08 IG7B08 IG5B08-IG7B08 IG5B08-IG7C08 IG7C08 61974 119 IG7C08 IG7B08 0.19%
IG5B11 IG7B11 IG5B11-IG7B11 IG5B11-IG7A11 IG7A11 47967 51 IG7A11 IG7B11 0.11%
IG5C03 IG7C03 IG5C03-IG7C03 IG5C03-IG7D03 IG7D03 49273 56 IG7D03 IG7C03 0.11%
IG5C07 IG7C07 IG5C07-IG7C07 IG5C07-IG7C06 IG7C06 45027 637 IG7C06 IG7C07 1.41%
IG5C08 IG7C08 IG5C08-IG7C08 IG5C08-IG7D08 IG7D08 67868 85 IG7D08 IG7C08 0.13%
IG5C11 IG7C11 IG5C11-IG7C11 IG5C11-IG7B11 IG7B11 57807 68 IG7B11 IG7C11 0.12%
IG5D07 IG7D07 IG5D07-IG7D07 IG5D07-IG7E07 IG7E07 38866 88 IG7E07 IG7D07 0.23%
IG5D07 IG7D07 IG5D07-IG7D07 IG5D07-IG7C08 IG7C08 38866 49 IG7C08 IG7D07 0.13%
IG5D08 IG7D08 IG5D08-IG7D08 IG5D08-IG7B10 IG7B10 53619 70 IG7B10 IG7D08 0.13%
IG5D08 IG7D08 IG5D08-IG7D08 IG5D08-IG7E08 IG7E08 53619 65 IG7E08 IG7D08 0.12%
IG5E07 IG7E07 IG5E07-IG7E07 IG5E07-IG7F07 IG7F07 57203 63 IG7F07 IG7E07 0.11%
IG5E08 IG7E08 IG5E08-IG7E08 IG5E08-IG7D07 IG7D07 72767 281 IG7D07 IG7E08 0.39%
IG5E09 IG7E09 IG5E09-IG7E09 IG5E09-IG7D09 IG7D09 58757 70 IG7D09 IG7E09 0.12%
IG5F03 IG7F03 IG5F03-IG7F03 IG5F03-IG7E03 IG7E03 54811 78 IG7E03 IG7F03 0.14%
IG5F06 IG7F06 IG5F06-IG7F06 IG5F06-IG7G10 IG7G10 50348 60 IG7G10 IG7F06 0.12%
IG5F08 IG7F08 IG5F08-IG7F08 IG5F08-IG7G08 IG7G08 67091 167 IG7G08 IG7F08 0.25%
IG5G12 IG7G12 IG5G12-IG7G12 IG5G12-IG7C01 IG7C01 40234 64 IG7C01 IG7G12 0.16%
IG5H03 IG7H03 IG5H03-IG7H03 IG5H03-IG7H02 IG7H02 48832 58 IG7H02 IG7H03 0.12%
IG5H06 IG7H06 IG5H06-IG7H06 IG5H06-IG7A11 IG7A11 42784 62 IG7A11 IG7H06 0.14%
IG5H07 IG7H07 IG5H07-IG7H07 IG5H07-IG7A08 IG7A08 36410 88 IG7A08 IG7H07 0.24%
IG5H11 IG7H11 IG5H11-IG7H11 IG5H11-IG7E11 IG7E11 32519 50 IG7E11 IG7H11 0.15%
It is for statistical analysis to table 8 and 9 data of table progress first time quality inspection result, available IG7A01-IG5A01's Relevant information, as shown in table 10 and Fig. 3；In addition, the dirt of available IG7 and IG5 for statistical analysis to 96 pairs of Tag primers Contaminate the distribution map (Fig. 6) of the pollution accounting of accounting hotspot graph (Fig. 4 and Fig. 5) and IG7 and IG；Summarize the conclusion obtained Are as follows: 1) sequence that this combination of IG7A01-IG5A01 measures is few, and the sequence containing IG7A01 only has 96, the sequence containing IG5A01 Column also only have 26, well below quality inspection need at least 5000 and accounting > 0.2% requirement；2) due to combination of the above Sequence it is few, the combination uniquely measured is forbidden combination again, thus pollution ratio it is very high；3) synthesis apparently, corresponds to This hole IG7A01-IG5A01 is problematic, no matter is all desirable from ordered sequence number or for contaminated possibility Replacement.
Table 10
Since this hole IG7A01-IG5A01 is problematic, this 2 strip label of separately synthesized IG7A01 and IG5A01 again The primer that primer dissolution recombines is put into the corresponding aperture of new deep-well plates in proportion to normal concentration, is removed original All liq remaining in the original mother plate of quality inspection failure is transferred to one piece of new deep-well plates by the corresponding hole IG7A01-IG5A01 Interior corresponding position, molecule plate carries out pollution Quality Control detection again；Second of Analysis of quality control report result as shown in table 11 and table 12, The case where table 11 and table 12 only list pollution.
Table 11 is directed to the sequencing result of the end IG7 Index
Query It is expected that compound object It is expected that combining Unexpected combination Unexpected compound object Total sequence item number Unexpected composite sequence item number Pollution sources It is contaminated Pollute accounting
IG7A01 IG5A01 IG7A01-IG5A01 IG7A01-IG5B01 IG5B01 490179 579 IG5B01 IG5A01 0.12%
IG7A08 IG5A08 IG7A08-IG5A08 IG7A08-IG5H07 IG5H07 244997 300 IG5H07 IG5A08 0.12%
IG7A11 IG5A11 IG7A11-IG5A11 IG7A11-IG5B11 IG5B11 398075 580 IG5B11 IG5A11 0.15%
IG7B02 IG5B02 IG7B02-IG5B02 IG7B02-IG5A03 IG5A03 285357 358 IG5A03 IG5B02 0.13%
IG7B10 IG5B10 IG7B10-IG5B10 IG7B10-IG5D08 IG5D08 262786 435 IG5D08 IG5B10 0.17%
IG7B11 IG5B11 IG7B11-IG5B11 IG7B11-IG5C11 IG5C11 336262 664 IG5C11 IG5B11 0.20%
IG7C06 IG5C06 IG7C06-IG5C06 IG7C06-IG5C07 IG5C07 345406 4253 IG5C07 IG5C06 1.23%
IG7C08 IG5C08 IG7C08-IG5C08 IG7C08-IG5B08 IG5B08 306713 460 IG5B08 IG5C08 0.15%
IG7C09 IG5C09 IG7C09-IG5C09 IG7C09-IG5D09 IG5D09 233352 284 IG5D09 IG5C09 0.12%
IG7C11 IG5C11 IG7C11-IG5C11 IG7C11-IG5D11 IG5D11 343633 744 IG5D11 IG5C11 0.22%
IG7D01 IG5D01 IG7D01-IG5D01 IG7D01-IG5C01 IG5C01 260626 313 IG5C01 IG5D01 0.12%
IG7D03 IG5D03 IG7D03-IG5D03 IG7D03-IG5C03 IG5C03 230602 238 IG5C03 IG5D03 0.10%
IG7D03 IG5D03 IG7D03-IG5D03 IG7D03-IG5E03 IG5E03 230602 264 IG5E03 IG5D03 0.11%
IG7D07 IG5D07 IG7D07-IG5D07 IG7D07-IG5E08 IG5E08 243561 1585 IG5E08 IG5D07 0.65%
IG7D07 IG5D07 IG7D07-IG5D07 IG7D07-IG5C07 IG5C07 243561 255 IG5C07 IG5D07 0.10%
IG7D08 IG5D08 IG7D08-IG5D08 IG7D08-IG5C08 IG5C08 316348 448 IG5C08 IG5D08 0.14%
IG7D09 IG5D09 IG7D09-IG5D09 IG7D09-IG5E09 IG5E09 351153 672 IG5E09 IG5D09 0.19%
IG7E07 IG5E07 IG7E07-IG5E07 IG7E07-IG5D07 IG5D07 314916 468 IG5D07 IG5E07 0.15%
IG7E08 IG5E08 IG7E08-IG5E08 IG7E08-IG5D08 IG5D08 313695 328 IG5D08 IG5E08 0.10%
IG7E08 IG5E08 IG7E08-IG5E08 IG7E08-IG5A11 IG5A11 313695 318 IG5A11 IG5E08 0.10%
IG7E12 IG5E12 IG7E12-IG5E12 IG7E12-IG5D12 IG5D12 189902 195 IG5D12 IG5E12 0.10%
IG7G01 IG5G01 IG7G01-IG5G01 IG7G01-IG5F01 IG5F01 271347 362 IG5F01 IG5G01 0.13%
IG7G08 IG5G08 IG7G08-IG5G08 IG7G08-IG5F08 IG5F08 202711 733 IG5F08 IG5G08 0.36%
IG7G09 IG5G09 IG7G09-IG5G09 IG7G09-IG5H09 IG5H09 268926 317 IG5H09 IG5G09 0.12%
IG7G10 IG5G10 IG7G10-IG5G10 IG7G10-IG5F06 IG5F06 363743 533 IG5F06 IG5G10 0.15%
IG7G10 IG5G10 IG7G10-IG5G10 IG7G10-IG5F10 IG5F10 363743 510 IG5F10 IG5G10 0.14%
IG7H02 IG5H02 IG7H02-IG5H02 IG7H02-IG5H03 IG5H03 237964 294 IG5H03 IG5H02 0.12%
IG7H07 IG5H07 IG7H07-IG5H07 IG7H07-IG5G07 IG5G07 240529 355 IG5G07 IG5H07 0.15%
IG7H08 IG5H08 IG7H08-IG5H08 IG7H08-IG5A09 IG5A09 121660 253 IG5A09 IG5H08 0.21%
Table 12 is directed to the sequencing result of the end IG5 Index
After primer replacement operation, cross contamination is not present in IG7A01-IG5A01, and 96 pairs of Tag primer indices are all Meet quality testing standard.
In addition, the present embodiment is also compared first time Analysis of quality control and second of Analysis of quality control, as a result such as table 13, figure Shown in shown in 7 (being compared analysis to IG7 Tag primer) and Fig. 8 (being compared analysis to IG5 Tag primer), it can be seen that The reproducibility of Analysis of quality control or good twice, it is seen that the stabilization of quality control method of the present invention is preferable.
Table 13
Embodiment 3
The present embodiment carries out quality inspection with 96 pairs of library labels, for statistical analysis to first time quality inspection result, available The relevant information of one pair of them Tag primer, as shown in Figure 9；In addition, it is for statistical analysis to 96 pairs of Tag primers, it is available The distribution map (Figure 12) of the pollution accounting of the pollution accounting hotspot graph (Figure 10 and Figure 11) and IG7 and IG of IG7 and IG5；Summarize The conclusion obtained:, to Tag primer through first time Analysis of quality control, 96 pairs of Tag primers meet index for this.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and Range.

Claims (10)

1. a kind of for detecting the quality control method of unique both-end library tag combination, which comprises the following steps:
S1) using library tag standards product and gDNA standard items as raw material, gDNA of the building with unique both-end library tag combination The library built is carried out upper machine sequencing, and reads library sequence label by library；
S2 first time Analysis of quality control) is carried out to library sequence label, the index of Analysis of quality control includes following items: maximum unilateral side Label pollution accounting≤2.5%, maximum tag combination pollute accounting≤0.01%, every group of exemplar sequence item number >=5000 Item, all tag combinations mix accounting coefficient of variation≤0.5, Compositive sequence percent of pass >=97%, and every group of exemplar sequence accounts for Than >=0.2/ library tag combination logarithm, the label accounting that unilateral side is greater than 1% pollution answers≤10%；
S3) if step S2) Analysis of quality control show index do not meet, recombine the library label for not meeting Quality Control requirement；It presses It is former with the satisfactory library label of library label, first time Analysis of quality control and gDNA that recombine according to step S1) method Material, gDNA library of the building with unique both-end library tag combination, re-starts upper machine for the library built and is sequenced, and read Take library sequence label；
S4 second of Analysis of quality control) is carried out to library sequence label, until all library labels meet the index of Analysis of quality control；
In the parameter of Analysis of quality control, the uniqueness both-end library tag combination is by upstream library label and downstream library label Composition, upstream library label are referred to as IG5, and IG5 is included as A and B；Downstream library label is referred to as IG7, IG7 packet Containing a and b；Matching and correctly uniqueness both-end library tag combination are A-a and B-b；Unmatched uniqueness both-end library label Group is combined into A-b and B-a；By analyzing the available respective sequence item number of combination of the above after each sequencing reaction；
The unilateral side label pollution accounting is the cross contamination ratio occurred between group interior label, and pollution is only possible to occur in group It is interior, i.e., it is polluted in IG5 group or/and in IG7 group；
When any cross contamination does not occur in process of production for a of IG7, for the A of IG5, wherein the pollution accounting containing B= Sequence item number containing B-a/all sequence item numbers containing a,
When any cross contamination does not occur in process of production for the A of IG5, for a of IG7, wherein the pollution accounting containing b= Contain A-b sequence item number/all sequence item numbers containing A；
When B pollutes A and b pollution a, then B-b tag combination pollutes accounting=(sequence item number containing B-a/all sequences containing a Column item number) × (containing A-b sequence item number/all sequence item numbers containing A)；
When any cross contamination does not occur in process of production for the b of IG7, for the B of IG5, wherein the pollution accounting containing A= Sequence item number containing A-b/all sequence item numbers containing b,
When any cross contamination does not occur in process of production for the B of IG5, for the b of IG7, wherein the pollution accounting containing a= Contain B-a sequence item number/all sequence item numbers containing B；
When A pollutes B and a pollution b, then A-a tag combination pollutes accounting=(sequence item number containing A-b/all sequences containing b Column item number) × (containing B-a sequence item number/all sequence item numbers containing B)；
Every group of exemplar sequence item number is to contain A-a by filtered every group correct matched sequence item number of system Sequence item number or sequence item number containing B-b；
All tag combination mixing accounting coefficient of variation are to be existed by filtered every group correct matched sequence item number of system Pass through variance of proportion coefficient in the filtered total correct sequence item number of pairing of system；
The Compositive sequence percent of pass be sequencing reaction after by system it is filtered it is correct pairing and ordered sequence total number Account for the ratio of all sequences total number after filtering by system；
Every group of exemplar sequence accounting is that the sequence item number correctly matched by filtered every group of system accounts for pass through and is The ratio of total sequence after system filtering；
The unilateral label accounting for being greater than 1% pollution are as follows: in the label of upstream library, pollution ratio is greater than 1% library label The ratio of the number total library number of tags of Zhan；And in the label of downstream library, pollution ratio is greater than the 1% library total library number of tags Zhan The ratio of number of tags.
2. quality control method as described in claim 1, which is characterized in that the step S1) successively the following steps are included: gDNA is marked Quasi- product prepare, gDNA fragmentation, and end is repaired, connector connection, the purifying of connector connection product, and amplified library expands the pure of library Change, the quality inspection of purified library, machine sequencing in the detection of purified library clip size and library.
3. quality control method as described in claim 1, which is characterized in that it is described uniqueness both-end library tag combination by IG5 group with IG7 group composition, the sequence of the respective library label between the Hamming distance >=3, IG5 and IG7 group of the library label in group of IG5 and IG7 Column Hamming distance >=2.
4. quality control method as claimed in claim 3, which is characterized in that library label is purified by high performance liquid chromatography And molecular weight is confirmed by mass spectral analysis, it is desirable that purity >=85%.
5. quality control method as described in claim 1, which is characterized in that the uniqueness both-end library tag combination is by 96 pairs of libraries Label composition, i.e., have 96 upstreams library label, there is 96 downstreams library label in IG7 group in IG5 group, corresponds；Every group Exemplar sequence accounting is then accordingly adjusted to >=0.2%.
6. quality control method as described in claim 1, which is characterized in that the uniqueness both-end library tag combination is by 48 pairs of libraries Label composition, i.e., have 48 upstreams library label, there is 48 downstreams library label in IG7 group in IG5 group, corresponds；Every group Exemplar sequence accounting is then accordingly adjusted to >=0.4%.
7. quality control method as described in claim 1, which is characterized in that when unique both-end library tag combination is by 192 pairs of libraries Label composition, i.e., have 192 upstreams library label, there is 192 downstream library labels in IG7 group in IG5 group, corresponds, every group Exemplar sequence accounting is then accordingly adjusted to >=0.1%.
8. quality control method as described in claim 1, which is characterized in that when unique both-end library tag combination is by 288 pairs of libraries Label composition, i.e., have 288 upstreams library label, there is 288 downstream library labels in IG7 group in IG5 group, corresponds, every group Exemplar sequence accounting is then accordingly adjusted to >=0.07%.
9. quality control method as described in claim 1, which is characterized in that when unique both-end library tag combination is by 384 pairs of libraries Label composition, i.e., have 384 upstreams library label, there is 384 downstream library labels in IG7 group in IG5 group, corresponds, every group Exemplar sequence accounting is then accordingly adjusted to >=0.05%.
10. the quality control method as described in claim 1~9 is applied in sample sequence measurement.
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CN111910258A (en) * 2020-08-19 2020-11-10 纳昂达(南京)生物科技有限公司 Paired-end library tag composition and application thereof in MGI sequencing platform
CN115197999A (en) * 2022-07-15 2022-10-18 纳昂达(南京)生物科技有限公司 Method and device for synthesizing crosstalk by quality control double-end unique tag connector

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