CN107217102B - A kind of the primer sequence group and method in quickly and efficiently quality inspection library - Google Patents
A kind of the primer sequence group and method in quickly and efficiently quality inspection library Download PDFInfo
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Abstract
The invention discloses a kind of primer sequence groups and method quickly and efficiently carrying out Quality Control to library.The primer sets are NGS Lqc L1 F, NGS Lqc L1 R；NGS Lqc M1 F, NGS Lqc M1 R；NGS Lqc H1 F, NGS Lqc H1 R.The present inventor is high, neutralization 3 pairs of primers of low three regions design by the G/C content to genome, as a control group with genome, Qpcr experiments are carried out to the library built up and genome, the quality in library is judged with 1 relationship according to the ratio of low GC amplification times/middle GC amplification times.
Description
Technical field
The invention belongs to high-flux sequence fields, are more particularly to a kind of primer quickly and efficiently carrying out Quality Control to library
Sequence group and method.
Background technology
With the development of two generation sequencing technologies, conventional liquid phase capture flow is divided into：It interrupts, build library and capture.This process
It from the interrupting of genome, captures and the sequencing of upper machine and data analysis at least need 6-7 days time, at present in addition to Qbit and 2100 point
It is other that library concentration and clip size are carried out outside Quality Control, without a kind of fast and effectively method can to build the quality of library result into
Row is judged.And the quality of Library Quality is of crucial importance sequencing result, in order to Library Quality sentence with imitating
Disconnected, to save time and sequencing cost, therefore the technology in rapid quality library is badly in need of in this field.
Invention content
The problem of for this field.The present inventor is the high, areas neutralization Di Sange by the G/C content to genome
3 pairs of primers are designed in domain, as a control group with genome, carry out Qpcr experiments to the library built up and genome, are expanded according to low GC
Number/ratio of middle GC amplification times is doubled to judge the quality in library with 1 relationship.
Therefore, in a first aspect, the present invention provides a kind of quickly to the method for library progress Quality Control, the method packet
It includes：
1) by NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1 this 3 pairs of primers interrupt to reference to DNA and to be measured
Library carries out Qpcr experiments, detects 6 ct value ct1-ct6 that each pair of primer corresponds to reference DNA and library to be measured, wherein：
Ct1 refer to NGS-Lqc-L1 this to primer pair with reference to DNA carry out Qpcr amplifications after ct values；
Ct4 refer to NGS-Lqc-L1 this to primer pair library to be measured carry out Qpcr amplifications after ct values；
Ct2 refer to NGS-Lqc-M1 this to primer pair with reference to DNA carry out Qpcr amplifications after ct values；
Ct5 refer to NGS-Lqc-M1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
Ct3 refer to NGS-Lqc-H1 this to primer pair with reference to DNA carry out Qpcr amplifications after ct values；
Ct6 refer to NGS-Lqc-H1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
2) it is low that the low GC amplification times/middle GC amplification times of M=are calculated, N=high GC amplification times/middle GC amplification times
Value, calculation formula M=2(ct1-ct4)/2(ct2-ct5), N=2(ct3-ct6)/2(ct2-ct5),
3) library homogeneity to be measured judges：M values are 0.3 or more, preferably 0.37 or more, and closer to 1, indicate to be measured
The homogeneity in library is better；N simultaneously>1.
In one embodiment, it is preferably genomic DNA with reference to DNA.
In one embodiment, it is 150bp-200bp to the clip size after being interrupted with reference to DNA to interrupt.
In second aspect, the present invention provides a kind of method quickly carrying out Quality Control to library, the method includes：
1) Qpcr experiments, detection are carried out by this 3 couple of NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1 library to be measured
Each pair of primer corresponds to 3 ct value ct4-ct6 in library to be measured,
Ct4 refer to NGS-Lqc-L1 this to primer pair library to be measured carry out Qpcr amplifications after ct values；
Ct5 refer to NGS-Lqc-M1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
Ct6 refer to NGS-Lqc-H1 this to primer pair to detect library carry out Qpcr amplifications after ct values；；
2) it is low that the low GC amplification times/middle GC amplification times of M=are calculated, N=high GC amplification times/middle GC amplification times
Value, calculation formula M=2(ct1-ct4)/2(ct2-ct5), N=2(ct3-ct6)/2(ct2-ct5), wherein Ct1, Ct2 and Ct3 is about respectively
21, about 21 and about 18,
3) library homogeneity to be measured judges：M values are 0.37 or more, and closer to 1, homogeneity is better；N simultaneously>1.
In one embodiment, the library clip size of building in library to be measured is 150bp-200bp.
In the third aspect, the present invention also provides a kind of primer sequence groups in quickly and efficiently quality inspection library：
NGS-Lqc-L1-F, NGS-Lqc-L1-R；NGS-Lqc-M1-F, NGS-Lqc-M1-R；NGS-Lqc-H1-F, NGS-
Lqc-H1-R。
The advantages of method and primer sequence group of the present invention：
1) content for designing low GC is 20%, and middle G/C content is 50%, high GC content 80%, by every on genome
It is analyzed every the G/C content of 100 bases, 100 pairs of primers is separately designed to high, low three regions of neutralization, pass through sequence alignment
It was found that high, normal, basic respectively there are 3 pairs of primer extension products single, NGS-Lqc-L1-F, NGS-Lqc-L1-R is found through experiments that；NGS-
Lqc-M1-F、NGS-Lqc-M1-R；There is preferable specificity with NGS-Lqc-H1-F, NGS-Lqc-H1-R primer.
2) Library Quality judgment criteria formula is dexterously devised, M values illustrate that the homogeneity in library is better closer to 1.
Specific implementation mode
In the present invention, it is preferred to using the method for first aspect present invention, it is real Qpcr will to be carried out with reference to DNA and library to be measured
It tests, can effectively avoid the deviation of experiment in this way.For saving reason or not including the feelings with reference to DNA for other reasons
Condition can also use the experience that obtains of inventor's long-term experiment with reference to the Ct values of DNA, i.e., Ct1, Ct2 and Ct3 be about 21 respectively,
About 21 and about 18, here " about " refer to that a reference value floats up and down 5%.
The present inventor develops a kind of method in rapid quality library, by genome, library homogeneity 20% with
Upper and 20% high, normal, basic 3 regions below separately design 3 pairs of primers, then carry out real-time quantitative PCR experiment, calculate separately out
After amplification times of high, normal, basic 3 regions for genome ct values, the size of M and N are calculated, experimental result is found
For homogeneity is in 20% or more library, for M sizes 0.37 or more, and for genome, ratio is about 1,
And N>1, which is because, Qpcr polymerases have Preference to the amplification in the high regions GC, it is difficult to find rule.Therefore pass through ratio
Compared with the relationship of M and 1, Quality Control is carried out to Library Quality.
Embodiment
1) following primer screening is carried out：To genome G/C content be respectively 20%, 50% and 80% three regions into
Then row design of primers 100 finds that this 3 regions respectively have 3 pairs of primer extension products single (described to primer by sequence alignment
1) 9 pairs of primers are shown in Table, carry out PCR experiment with this 9 pairs of primers respectively, as a result, it has been found that, NGS-Lqc-L1-F, NGS-Lqc-L1-R；
NGS-Lqc-M1-F, NGS-Lqc-M1-R；And NGS-Lqc-H1-F, NGS-Lqc-H1-R primer have preferable specificity.
Table 1：9 pairs of primers
Form three primer sequence table groups：
(1) SEQ ID NO.1 and SEQ ID NO.4；SEQ ID NO.2 and SEQ ID NO.5；SEQ ID NO.3 and SEQ
ID NO.6；
(2) SEQ ID NO.7 and SEQ ID NO.10；SEQ ID NO.11 and SEQ ID NO.5；SEQ ID NO.9 and
SEQ ID NO.12；
(3) SEQ ID NO.13 and SEQ ID NO.16；SEQ ID NO.17 and SEQ ID NO.5；SEQ IDNO.15 and
SEQ ID NO.18。
2) pass through NGS-Lqc-L1 (i.e. SEQ ID NO.1 and SEQ ID NO.4), NGS-Lqc-M1 (i.e. SEQ IDNO.2
With SEQ ID NO.5) and NGS-Lqc-H1 (i.e. SEQ ID NO.3 and SEQ ID NO.6) this 3 couples of primer pair genome gDNA,
It is real that 117R01129,117R01134 and library 117R01128-LiB, 117R01129-LiB and 117R01134-LiB carry out Qpcr
It tests.Genome gDNA, 117R01129 and 117R01134 are extracted by gene, are interrupted, and instrument Bioruptor is interrupted by ultrasound
Pico, after SAPMAC method instrument temperature is down to 4 DEG C, arrange parameter ON 30s, OFF 30s are 1 cycle, and every 10 cycle is a wheel,
3 wheels are carried out altogether, sample is placed on oscillator after every group and is mixed well, and next round is carried out after of short duration centrifugation and is interrupted, is interrupted
Clip size afterwards is 150bp-200bp.Library 117R01128-LiB, 117R01129-LiB and 117R01134-LiB to be measured
To build library process as follows：It by gene extraction, interrupts, instrument Bioruptor Pico is interrupted by ultrasound, wait for SAPMAC method instrument temperature
After being down to 4 DEG C, arrange parameter ON 30s, OFF30s are 1 and recycle, and every 10 cycle is a wheel, carry out 3 altogether and take turns, after every group
Sample is placed on oscillator and is mixed well, next round is carried out after of short duration centrifugation and is interrupted, the clip size after interrupting is 150bp-
200bp, air exercise part section carry out end reparation and add A, connection, PCR amplification, capture 117R01128-LiB, 117R01129-
This 3 libraries LiB and 117R01134-LiB.The specific design scheme of the present embodiment is as follows：
3 pipes are respectively configured and contain NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1 this 3 pairs of primers, 2 ×
60 parts of mixtures of iTaqTMuniversal SYBR Green Supermix after mixing, respectively add this 3 pipe mixture
Enter into Qpcr plates, 18ul is added per hole, often pipe is added 18 holes, totally 54 holes, later 2ul, a concentration of 5ng/ul's
GDNA, 117R01129,117R01134 and library 117R01128-LiB, 117R01129-LiB and 117R01134-LiB this 6
A template is added separately in the hole containing this 3 pairs of primers of NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1, the same mould
The corresponding same pair of primers of plate needs to do 3 multiple holes, and the result for repeating experiment uses average value below.
3) according to the sample being added in each hole, Qpcr response procedures are set, the ct values detection in each hole are carried out, to same
Correspondence in a sample ct value different with 3 of pair of primers is averaged, as 6 samples correspond to NGS-Lqc-L1,
The ct values of this 3 pairs of primers of NGS-Lqc-M1 and NGS-Lqc-H1.
4) data analysis：M=2(the low GC samples to be tested of the low GC genomes-ct of ct)/2(GC samples to be tested in GC genomes-ct in ct), N=2(ct The high GC samples to be tested of high gene group-ct)/2(GC samples to be tested in GC genomes-ct in ct)
Ct values：Refer to the PCR corresponding when detectable fluorescent emission statistically significantly can be generated on baseline to follow
Number of rings.Baseline range from the 3rd cycle play Ct values before 3 cycles terminate, 10 times of fluorescence intensity standard deviation in baseline range
As threshold value, and the intersection point of the horizontal line where threshold value and PCR amplification curve is exactly Ct values, between generally taking 3-15 cycle, Ct
Value and the relationship of starting template with the logarithm of the starting copy number of the template studies have shown that there is linear pass in the Ct values of each template
System, starting copy number is more, and Ct values are smaller.
The low GC genomes of Ct refer to NGS-Lqc-L1 this to primer pair genome carry out Qpcr amplifications after ct values；
The libraries to be measured the low GC of Ct refer to NGS-Lqc-L1 this to primer pair sample to be tested carry out Qpcr amplifications after ct values；
In Ct GC genomes refer to NGS-Lqc-M1 this to primer pair genome carry out Qpcr amplifications after ct values；
In Ct the libraries to be measured GC refer to NGS-Lqc-M1 this to primer pair to detect sample carry out Qpcr amplifications after ct
Value；
Ct high GC genomes refer to NGS-Lqc-H1 this to primer pair genome carry out Qpcr amplifications after ct values；
The libraries to be measured Ct high GC refer to NGS-Lqc-H1 this to primer pair to detect sample carry out Qpcr amplifications after ct
Value；
Genome refers to that gDNA, gDNA are by buying in the standard items of promega companies.
Sample to be tested refers to genome 117R01129,117R01134 and corresponding library 117R01129-LiB,
This 5 samples of 117R01134-LiB and independent library 117R01128-LiB；117R01129、117R01134、117R01128-
LiB, 117R01129-LiB and 117R01134-LiB both from different healthy volunteers leucocyte genome, before pressing
The method that text provides is prepared.
To genome gDNA, 117R01129,117R01134 and corresponding library 117R01129-LiB, 117R01134-LiB
Independent library 117R01128-LiB, by NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1 this 3 pairs of primer pairs this 6
Sample carries out Qpcr experiments respectively, and multiple holes number is 3.
To H1, M1 and L1, this 3 pairs of primers are configured to mixture, and system is according to sample size × 3+2=20.
Reaction system is as follows：
Qpcr amplifications are carried out, response procedures are as follows：
95℃ 3min
40 cycles：
95℃ 5s
62℃ 30s
Read fluorescence signal
After reaction, export Ct values are for statistical analysis.
Library information：
| Number | The target area ratio of 10% depth of mean depth both sides |
| 117R01128-LiB | 32.98% |
| 117R01129-LiB | 23.5% |
| 117R01134-LiB | 7.53% |
The target area ratio of 10% depth of mean depth both sides：The homogeneity of sequencing data is represented, ratio is higher, explanation
The homogeneity in library is higher, illustrates in library containing there are many different types of segments.By upper table as can be seen that 117R01134-
The target area ratio of 10% depth of mean depth both sides in the libraries LiB is 7.53%, belongs to unqualified library, as poor text
Library.This ratio generally at least requires, 15% or more, just to belong to qualified library, tightened up to take>About 20% value.
Find that for sequencing homogeneity is in 23.5% or more library, M is in 0.369078601-1 by experimental result
Within, and N>1, this is because iTaqTM universal SYBR Green Supermix have partially the amplification in the high regions GC
Good property, rule unobvious.So the foundation that can be judged using M as library quality, when ratio is closer to 1, illustrate the equal of library
One property is higher, and sequencing quality is higher.Based on above-mentioned, M values correspond to 23.5% homogeneity about 0.37.For>About 20%
Homogeneity value takes M values to be greater than about 0.3.
SEQUENCE LISTING
<110>Ai Jitaikang biotechnologies（Beijing）Co., Ltd
<120>A kind of the primer sequence group and method in quickly and efficiently quality inspection library
<130> CP20170651
<160> 18
<170> PatentIn version 3.5
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Claims (6)
1. a kind of method that Quality Control quickly being carried out to library, the method includes：
1) the reference DNA interrupted by NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1 this 3 pairs of primer pairs and library to be measured
Qpcr experiments are carried out, 6 ct value ct1-ct6 that each pair of primer corresponds to reference DNA and library to be measured are detected, wherein：
Ct1 refer to NGS-Lqc-L1 this to primer pair with reference to DNA carry out Qpcr amplifications after ct values；
Ct4 refer to NGS-Lqc-L1 this to primer pair library to be measured carry out Qpcr amplifications after ct values；
Ct2 refer to NGS-Lqc-M1 this to primer pair with reference to DNA carry out Qpcr amplifications after ct values；
Ct5 refer to NGS-Lqc-M1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
Ct3 refer to NGS-Lqc-H1 this to primer pair with reference to DNA carry out Qpcr amplifications after ct values；
Ct6 refer to NGS-Lqc-H1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
The NGS-Lqc-L1 is SEQ ID NO.1 and SEQ ID NO.4；The NGS-Lqc-M1 is SEQ ID NO.2 and SEQ
ID NO.5；The NGS-Lqc-H1 is SEQ ID NO.3 and SEQ ID NO.6；
2) it is low that the low GC amplification times/middle GC amplification times of M=are calculated, the value of N=high GC amplification times/middle GC amplification times, meter
Calculation formula is M=2(ct1-ct4)/2(ct2-ct5), N=2(ct3-ct6)/2(ct2-ct5),
3) library homogeneity to be measured judges：M values indicate that the homogeneity in library to be measured is better 0.3 or more, and closer to 1；Together
When N>1.
2. method of claim 1, in 3), M values are 0.37 or more.
3. method of claim 1, described to be preferably genomic DNA with reference to DNA.
4. method of claim 1, it is 150bp-200bp to the clip size after being interrupted with reference to DNA to interrupt.
5. a kind of method that Quality Control quickly being carried out to library, the method includes：
1) Qpcr experiments are carried out by this 3 couple of NGS-Lqc-L1, NGS-Lqc-M1 and NGS-Lqc-H1 library to be measured, detection is each pair of
Primer corresponds to 3 ct value ct4-ct6 in library to be measured,
Ct4 refer to NGS-Lqc-L1 this to primer pair library to be measured carry out Qpcr amplifications after ct values；
Ct5 refer to NGS-Lqc-M1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
Ct6 refer to NGS-Lqc-H1 this to primer pair to detect library carry out Qpcr amplifications after ct values；
The NGS-Lqc-L1 is SEQ ID NO.1 and SEQ ID NO.4；The NGS-Lqc-M1 is SEQ ID NO.2 and SEQ
ID NO.5；The NGS-Lqc-H1 is SEQ ID NO.3 and SEQ ID NO.6；
2) it is low that the low GC amplification times/middle GC amplification times of M=are calculated, the value of N=high GC amplification times/middle GC amplification times, meter
Calculation formula is M=2(ct1-ct4)/2(ct2-ct5), N=2(ct3-ct6)/2(ct2-ct5), wherein Ct1, Ct2 and Ct3 is about 21, about 21 respectively
About 18；
3) library homogeneity to be measured judges：M values are 0.37 or more, and closer to 1, homogeneity is better；N simultaneously>1.
6. a kind of primer sequence group in quickly and efficiently quality inspection library：
The primer sequence group includes SEQ ID NO.1 and SEQ ID NO.4；SEQ ID NO.2 and SEQ ID NO.5；SEQ
ID NO.3 and SEQ ID NO.6.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106283200A (en) * | 2016-09-03 | 2017-01-04 | 艾吉泰康生物科技(北京)有限公司 | A kind of library constructing method improving amplicon literature data homogeneity |
| CN106434924A (en) * | 2016-09-29 | 2017-02-22 | 北京全式金生物技术有限公司 | Kit and method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106283200A (en) * | 2016-09-03 | 2017-01-04 | 艾吉泰康生物科技(北京)有限公司 | A kind of library constructing method improving amplicon literature data homogeneity |
| CN106434924A (en) * | 2016-09-29 | 2017-02-22 | 北京全式金生物技术有限公司 | Kit and method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction) |
Non-Patent Citations (2)
| Title |
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| Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation;Jenifer Dang;《INTERNATIONAL JOURNAL OF ONCOLOGY》;20161231;第49卷;第1755-1765页 * |
| 山羊卵巢颗粒细胞差异表达基因消减文库的建立;庞训胜等;《南京农业大学学报》;20101231;第33卷(第6期);第95-100页 * |
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