CN111088328A - Kit and detection method for high-throughput sequencing library quantitative detection - Google Patents
Kit and detection method for high-throughput sequencing library quantitative detection Download PDFInfo
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Abstract
The application discloses a kit and a detection method for high-throughput sequencing library quantitative detection. The kit comprises a detection primer and a detection probe designed aiming at a joint sequence of a high-throughput sequencing library to be detected; the upstream and downstream primers of the detection primer are sequences shown by Seq ID No.1 and 2, and the detection probe is a sequence shown by Seq ID No. 3; the 5 'end of the detection probe is modified by a fluorescent group, and the 3' end of the detection probe is modified by a fluorescent quenching group. The kit can realize absolute quantification of the NGS library by carrying out quantitative detection on the NGS library through a TaqMan probe method, and can more accurately obtain the actual concentration of the NGS library without correcting the size of a DNA fragment of the NGS library based on the QPCR quantitative detection of the existing dye method, so that the kit has important significance for high-throughput sequencing, can improve the sequencing quality and further obtains the optimal sequencing data result.
Description
Technical Field
The application relates to the technical field of high-throughput sequencing, in particular to a kit and a detection method for high-throughput sequencing library quantitative detection.
Background
The purpose of preparing a high-throughput sequencing library (NGS library) is to link specific linker sequences to fragmented nucleic acid fragments to be tested, so that they can be sequenced on a high-throughput sequencing platform. The quality of NGS libraries is critical to the quality of data produced by high throughput sequencing (NGS). Therefore, before the NGS library is subjected to on-machine sequencing, the concentration of the NGS library needs to be accurately determined, so that the appropriate on-machine sequencing concentration can be adjusted during on-machine sequencing, and finally, the optimal sequencing data result is obtained.
The quantitative detection of the NGS library QPCR method is a library which can be sequenced by using specific primers to quantify the complete library connected with the joints at the two ends in a sample, can eliminate the interference of a non-sequencable library with one end or two ends not connected with the joints, and is the preferred method for quantifying the library in the industry at present. At present, quantitative detection aiming at NGS library by QPCR method in the market is mainly based on dye method; the basic principle of the quantitative detection is that the detection result of the NGS library to be detected is compared with a standard curve prepared by a standard substance, relative quantification is carried out through the standard curve, and then the quantitative result is corrected according to the size difference between the fragments of the NGS library to be detected and the fragments of the standard substance. Theoretically, the existing quantitative detection of the NGS library by QPCR can accurately quantify the NGS library and provide accurate concentration information for the on-machine sequencing.
However, in practice, it often happens that after concentration measurement is performed according to the quantitative detection result of dye method QPCR, optimized on-machine sequencing concentration is adopted, but the finally obtained sequencing data have uneven quality. For example, for DNA samples from different sources, especially under the condition of different DNA sample fragment sizes, the NGS libraries constructed therefrom have inconsistent quality of finally obtained sequencing data, although the same dye method QPCR quantitative detection is adopted and the sequencing is performed by using the same sequencing platform.
Disclosure of Invention
The application aims to provide a novel kit and a detection method for high-throughput sequencing library quantitative detection.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the application discloses a kit for quantitative detection of a high-throughput sequencing library, which comprises a detection primer and a detection probe designed aiming at a joint sequence of the high-throughput sequencing library to be detected; wherein, the upstream primer of the detection primer is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, and the detection probe is a sequence shown by Seq ID No. 3;
Seq ID No.1：5’-AATGATACGGCGACCACCGA-3’
Seq ID No.2：5’-CAAGCAGAAGACGGCATACGA-3’
Seq ID No.3：5’-ACACGACGCTCTTCCGATCT-3’
the 5 'end of the detection probe is modified by a fluorescent group, and the 3' end of the detection probe is modified by a fluorescent quenching group.
It should be noted that, the high-throughput sequencing library quantitative detection kit of the present application can perform quantitative detection on the high-throughput sequencing library by using the TaqMan probe method, can realize absolute quantification of the high-throughput sequencing library, solves the problem of influence of the sizes of DNA fragments of different high-throughput sequencing libraries on library concentration determination, can more accurately obtain the actual concentration of the high-throughput sequencing library, performs high-throughput sequencing according to the high-throughput sequencing library concentration determined by the kit of the present application, can improve sequencing quality, and thus obtains an optimal sequencing data result.
Preferably, the fluorescent group used in the detection probe of the kit of the present application is FAM, and the fluorescence quenching group is MGB.
It should be noted that the FAM fluorophore and the MGB fluorescence quencher are only one implementation manner of the present application, and the fluorophores and quenchers used are selected according to the specifically used real-time fluorescence quantitative PCR instrument, which does not exclude that other fluorophores or quenchers may also be used, such as commonly used fluorophores such as TET, JOE, HEX, and VIC, and commonly used quenchers such as TAMRA and BHQ.
Preferably, the kit of the present application further comprises reagents for real-time fluorescent quantitative PCR detection by TaqMan probe method.
It is understood that the TaqMan probe method real-time fluorescence quantitative PCR detection reagent can be purchased in the market, and can also be assembled into the kit of the application for convenient use.
Preferably, the kits of the present application further comprise an absolute quantitative standard containing a known nucleic acid concentration.
The application also discloses a quantitative detection method of the high-throughput sequencing library, which comprises the step of carrying out real-time fluorescence quantitative PCR detection on the high-throughput sequencing library to be detected by adopting the kit.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the high-throughput sequencing library quantitative detection kit carries out quantitative detection on the high-throughput sequencing library through a TaqMan probe method, can realize absolute quantification of the high-throughput sequencing library, and compared with the existing dye method QPCR quantitative detection, the kit can obtain the actual concentration of the high-throughput sequencing library more accurately, and does not need to correct the DNA fragment size of the high-throughput sequencing library, so that the kit has important significance on high-throughput sequencing, can improve the sequencing quality, and further obtains the optimal sequencing data result.
Detailed Description
The inventors of the present application have analyzed that the reason for performing on-machine sequencing based on the concentration determined by the QPCR quantitative detection result of the conventional dye method is that the quality of sequencing data varies, and it is difficult to absolutely unify the sizes of DNA fragments of different high-throughput sequencing libraries.
The dye QPCR quantitative detection is to compare the fluorescence signal or Ct value of the high-throughput sequencing library to be detected with a standard curve drawn by a standard substance so as to realize relative quantification, namely, the concentration of the high-throughput sequencing library to be detected is reversely deduced according to the fluorescence signal or Ct value. The fluorescence signal or Ct value of the high-throughput sequencing library to be detected is related to the concentration thereof and the size of the DNA fragment of the high-throughput sequencing library; it can be understood that for the same number of molecules of DNA sample, the larger the fragment, the more the corresponding bound fluorochrome, the stronger the fluorescence signal obtained; therefore, the ordinary dye-process QPCR quantitative detection needs concentration correction according to the difference between the size of the DNA fragment of the high-throughput sequencing library and the size of the DNA fragment of the standard.
However, in the case of an abnormal high-throughput sequencing library with multimodal fragments, the sizes of the DNA fragments are not consistent, that is, there are a plurality of clusters with different sizes of the DNA fragments, so it is difficult to accurately correct the quantitative detection result of dye QPCR, which causes deviation between the concentration value of the quantitative detection and the actual concentration, and further causes the quality of sequencing data to be uneven.
Based on the above research and understanding, the present application inventively develops and provides a novel TaqMan probe detection-based high-throughput sequencing library quantitative detection kit, which comprises a detection primer and a detection probe designed for a linker sequence of a high-throughput sequencing library to be detected; the upstream primer of the detection primer is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, and the detection probe is a sequence shown by Seq ID No. 3.
When the kit is used for quantitative detection, one probe is cut off to generate a unit signal when one target DNA chain is generated, the signal intensity is in direct proportion to the number of DNA molecules combined with the probe and is irrelevant to the size of a DNA fragment; therefore, the kit of the application can be used for quantitative detection of the NGS library, so that the step of result correction can be omitted, and more accurate concentration results can be obtained.
The kit disclosed by the application adopts a TaqMan probe method to carry out absolute quantification of the NGS library, solves the problem that the result correction is carried out by using the DNA fragment of the high-throughput sequencing library in the quantitative detection of the dye QPCR method, and avoids the inaccurate detection result caused by the abnormal DNA fragment of the high-throughput sequencing library when the evaluation cannot be carried out.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
This example uses human leukocyte genomic dna (gdna) for high throughput sequencing library construction based on the Illumina sequencing platform. Specifically, a KAPAhyper Prep Kit (KK850407962363001) Kit is adopted to construct a high-throughput sequencing library for three human leukocyte gDNAs. The specific steps of the high-throughput sequencing library construction refer to the KAPAHyperPrep Kit application instruction, and the high-throughput sequencing libraries constructed by three human leukocyte gDNAs are respectively marked as library 1, library 2 and library 3.
Wherein, the fragmentation of the gDNA sample is carried out by using an ultrasonic disruptor Covaris M220, and the specific parameters are as follows: gDNA 500. mu.L i.e. 1000ng, temperature 20 ℃, Peak Power 50.0, Duty Factor 30.0, Cycle/Burst 200, time 300 sec.
Aiming at an adaptor sequence of an Illumina sequencing platform, a detection primer and a detection probe aiming at the adaptor sequence are designed in the example; the upstream primer of the detection primer is a sequence shown in Seq ID No.1, the downstream primer is a sequence shown in Seq ID No.2, and the detection probe is a sequence shown in Seq ID No.3, and the detection probe is used as a kit for detecting the concentration of the library in the embodiment;
Seq ID No.1：5’-AATGATACGGCGACCACCGA-3’
Seq ID No.2：5’-CAAGCAGAAGACGGCATACGA-3’
Seq ID No.3：5’-ACACGACGCTCTTCCGATCT-3’
the 5 'end of the detection probe is modified by a fluorescent group, and the 3' end of the detection probe is modified by a fluorescent quenching group.
The concentration of the libraries 1, 2 and 3 constructed in this example was determined using the above primers and probes, and the specific PCR amplification system and amplification conditions are shown in Table 1 and Table 2, respectively.
TABLE 1TaqMan probe method PCR amplification reaction System
| Components | Final concentration | Reagent volume of 1 reaction |
| ForwardPrimerP5(10μM) | 0.2μM | 0.2μL |
| ReversePrimerP7(10μM) | 0.2μM | 0.2μL |
| TaqManMGBProbe(10μM) | 0.2μM | 0.2μL |
| 2×TransStartProbeQPCRSuperMix | 1× | 5μL |
| PassiveReferenceDye(50×) | 1× | 0.2μL |
| ddH2O | / | 2.2μL |
| Total volume | / | 8μL |
TABLE 2TaqMan probe method PCR amplification reaction conditions
In Table 1, Forward Primer P5 (10. mu.M), i.e., the upstream Primer of the sequence shown in Seq ID No.1 at 10. mu.M, Reverse Primer P7 (10. mu.M), i.e., the downstream Primer of the sequence shown in Seq ID No.2 at 10. mu.M, and TaqMan MGB Probe (10. mu.M), i.e., the detection Probe of the sequence shown in Seq ID No.3 at 10. mu.M, were used. 2 × TransStart Probe QPCR SuperMix is a commercially available Probe method for quantifying the reaction Mix. The Passive Reference Dye (50X) can be added or not according to the brand model of the equipment.
For comparison, the same three libraries were subjected to quantitative detection by the dye method QPCR of KAPANGS library, which was referred to the recommendations of Illumina sequencing platform.
The results of the dye method detection of KAPANGS library by TaqMan probe method and comparison are shown in Table 3.
TABLE 3 results of concentration measurements of the three libraries
In this example, the in-machine sequencing was performed according to the results of different concentrations of the same library obtained by the two quantitative methods, i.e., the concentrations shown in table 3, in this example, a NovaSeq6000 sequencing platform was specifically used for sequencing, the in-machine sequencing step was referred to NovaSeq6000 sequencing guide published by the ministry of Illumina, the NovaSeq XP mode was used for in-machine sequencing, and the sequencing concentration was selected to be 1.0nM based on the results of the quality control concentration calculation.
The quality control results of the sequencing data are shown in Table 4.
TABLE 4 partial quality control results based on sequencing data for different assay concentrations
The results in table 4 show that on-machine sequencing based on the NGS library concentration quantitatively determined by the NGS library probe method of this example can obtain a higher amount of off-machine data of library sequencing, and the off-machine data Dup of library sequencing is significantly reduced. This shows that the quantitative detection of the NGS library by the probe method based on the detection primer and the detection probe of the embodiment can obtain the concentration of the NGS library more accurately and effectively, and the detection result does not need to be corrected, so that the operation is simpler and the result is more accurate compared with the conventional quantitative detection of QPCR by the dye method. The kit of the embodiment can accurately and effectively detect the concentration of the high-throughput sequencing library, particularly for abnormal library types with multimodal fragments and the like, under the condition that the size of the fragments cannot be accurately estimated, the kit and the detection method of the embodiment can still stably obtain the true concentration value of the high-throughput sequencing library sample, and effectively ensure the quality and quantity of the NGS sequencing data.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen haipraos medical examination laboratory
Shenzhen Shanpulos Biotech Co Ltd
<120> kit and detection method for high-throughput sequencing library quantitative detection
<130>19I29590
<160>3
<170>PatentIn version 3.3
<210>1
<211>20
<212>DNA
<213> Artificial sequence
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aatgatacgg cgaccaccga 20
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<212>DNA
<213> Artificial sequence
<400>2
caagcagaag acggcatacg a 21
<210>3
<211>20
<212>DNA
<213> Artificial sequence
<400>3
acacgacgct cttccgatct 20
Claims (5)
1. A kit for quantitative detection of a high throughput sequencing library, characterized by: the method comprises the steps of designing a detection primer and a detection probe aiming at a joint sequence of a high-throughput sequencing library to be detected; the upstream primer of the detection primer is a sequence shown by Seq ID No.1, the downstream primer is a sequence shown by Seq ID No.2, and the detection probe is a sequence shown by Seq ID No. 3;
Seq ID No.1：5’-AATGATACGGCGACCACCGA-3’
Seq ID No.2：5’-CAAGCAGAAGACGGCATACGA-3’
Seq ID No.3：5’-ACACGACGCTCTTCCGATCT-3’
the 5 'end of the detection probe is modified by a fluorescent group, and the 3' end of the detection probe is modified by a fluorescent quenching group.
2. The kit of claim 1, wherein: the fluorescent group is FAM, and the fluorescence quenching group is MGB.
3. The kit according to claim 1 or 2, characterized in that: also comprises a reagent for real-time fluorescent quantitative PCR detection by a TaqMan probe method.
4. The kit according to claim 1 or 2, characterized in that: absolute quantitative standards containing known nucleic acid concentrations are also included.
5. A quantitative detection method of a high-throughput sequencing library is characterized by comprising the following steps: comprises the TaqMan probe real-time fluorescent quantitative PCR detection of a high-throughput sequencing library to be detected by adopting the kit of any one of claims 1 to 4.
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| CN113186262A (en) * | 2021-04-09 | 2021-07-30 | 上海锐翌生物科技有限公司 | Method and kit for rapid quantification of MGI platform high-throughput sequencing library |
| CN113588392A (en) * | 2021-07-28 | 2021-11-02 | 北京金匙基因科技有限公司 | Quantitative sample mixing method for improving sequencing sample mixing uniformity |
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