CN117867086A - Standard substance for quantitative high-throughput sequencing library and preparation method and application thereof - Google Patents
Standard substance for quantitative high-throughput sequencing library and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of gene sequencing, in particular to a standard substance for quantitative use of a high-throughput sequencing library, a preparation method and application thereof. The high-throughput standard substance for sequencing library quantification is obtained by PCR amplification of template DNA comprising sequence insert fragments, and the sequence insert fragments and the standard substance for sequencing library quantification obtained by amplification thereof cannot be compared with human beings, common animals, plants and microorganisms, so that the pollution of related sequences of the standard substance for sequencing library quantification possibly caused by various reasons can be effectively avoided, and the accuracy of sequencing data result analysis of a sample of a library to be detected is improved. Meanwhile, the base ratio of the standard substance for quantitative sequencing library provided by the invention in the range of a plurality of sections is balanced, and when the standard substance for quantitative sequencing library is used for real-time fluorescence quantitative PCR, the amplification efficiency is high and stable, and the phenomenon that the amplification efficiency is poor or unstable due to the complex sequence of the standard substance for quantitative sequencing library is avoided.
Description
Technical Field
The invention relates to the technical field of gene sequencing, in particular to a standard substance for quantitative use of a high-throughput sequencing library, a preparation method and application thereof.
Background
The high-throughput sequencing technology can detect multiple genes and multiple targets simultaneously, and is widely used for detecting gene sequences of human beings and various other animals and plants and microorganisms. However, due to the high cost of high throughput sequencing, it is desirable to quantify the library sample to be tested prior to on-machine sequencing. Accurate quantitative analysis of the library is critical to obtaining high quality sequencing data. If the library quantification concentration is high, it may result in insufficient sequencing data yield; whereas lower quantitative concentrations may lead to low quality data and even sequencing failures; in addition, along with the improvement of the sequencing flux of the sequencer, a plurality of libraries are often combined together for sequencing, and the libraries with corresponding concentrations are mixed according to the requirement of sequencing data volume and the quantitative result so as to stably output the sample data volume of each library to be detected. Thus, accurate quantification of library concentrations is essential in high throughput sequencing technologies.
At present, the quantification of the library sample to be detected can be carried out by methods such as a fluorometer, real-time fluorescence quantitative PCR and the like. However, when the fluorometer is used for quantifying double-stranded DNA, it is impossible to distinguish between an ineffective library (i.e., a double-stranded DNA fragment that cannot be subjected to subsequent high-throughput sequencing) and an effective library (i.e., a library that contains the complete sequence structure of the library and can be subjected to subsequent high-throughput sequencing). And the real-time fluorescent quantitative PCR can be carried out by designing primers and probes aiming at the joint sequences at two ends of the complete structure library, and detecting the real-time fluorescent quantitative PCR by hydrolyzing the probes or directly carrying out the real-time fluorescent quantitative PCR by combining the primers and fluorescent dyes through joint design. Thus, the interference of the non-sequencable ineffective library of the single-end or double-end unconnected joint can be eliminated, so that the real-time fluorescence quantitative PCR method is used as a gold standard for library quantification, and the accuracy is highest.
When the concentration of a sample of a library to be detected is detected by real-time fluorescent quantitative PCR, a group of standard substances with known concentrations are needed to be used for manufacturing a standard curve, an X axis and a Y axis are respectively logarithmic values of the concentration (or the mass) of the standard substances, ct values of the standard substances in the fluorescent quantitative PCR, the Ct values and logarithms of the concentration (or the mass) of a template show a negative linear relationship, and a standard curve formula (Y=kX+b, wherein k and b are respectively the slope and intercept of the standard curve) can be obtained by straight line fitting. Substituting the Ct value of the library sample to be detected into a linear standard curve, and obtaining the concentration (or mass, unit consistent with that of the standard substance) of the library sample to be detected. It can be seen that the amplification efficiency and stability of the standard are critical to the accuracy of the quantitative results of the library samples to be tested. At present, in the field of high-throughput sequencing, a standard substance used when a real-time fluorescent quantitative PCR is adopted for quantifying a library sample to be detected is usually adopted as an existing library sample, a library with higher concentration is firstly obtained through PCR amplification, and fragment screening is sometimes carried out; then quantifying by an absorbance method or a fluorometer; subsequent dilutions resulted in a series of concentration gradients of library standards. The main problems of this approach are: the absorbance method or the fluorometer cannot distinguish between an invalid library and a valid library, so that the quantitative result is inaccurate; the existing library sample is a mixture, and DNA sequences contained in the library sample are complex in composition (are composed of a large number of DNA fragments with different base sequences and different lengths), so that standard substances among different batches have extremely large differences in sequence, length and the like, and the amplification efficiency of the standard substances is difficult to realize batch-to-batch consistency; in addition, the library standard may be degraded during long-term storage, resulting in deviation of the quantitative result due to degradation of the storage quality of the library standard. These problems can ultimately lead to large differences in the throughput of high throughput sequencing data from the expected, and poor uniformity between the sequencing data volumes of different libraries to be tested. In view of the foregoing, there is a need for a method that can rapidly, stably and accurately prepare a standard for quantitative use in high throughput sequencing libraries.
Disclosure of Invention
In order to solve the technical problems, the invention provides a standard substance for quantitative analysis of a high-throughput sequencing library, and a preparation method and application thereof, so that risks caused by inaccurate quantitative analysis of the library to be sequenced are effectively reduced.
Specifically, the technical scheme of the invention is as follows:
in a first aspect, the invention provides a standard for quantifying a high throughput sequencing library, which is obtained by PCR amplification of a template DNA comprising a sequence insert, wherein the nucleotide sequence of the sequence insert is shown as SEQ ID NO. 1.
Sequence inserts:
ACGGTAGTCACGCCTCGTATCTACGTCAGGATGAGACTAGTACAGATATACCTGCTGTCGGCAGAAGTGTCATAGGAGTCACTGAATTCGTATAGCCTAATCACATCCGTCTCCGTGCGGGACAGTCACCGACGGCGACTCATAGTCGCCTAGTCGATCAGATTGGTCTAAGTAGCTTATGAGAGTACACTCTGCGATCATTCAGCAGTGATCACTGATGCTCAGTGCAGCTAGCTATGCGGATCGTCACAGATACTAGCCAGCTGATCTCAGCTAGTCTAGTTGACGATCTAGACGTGC
the length of the sequence insert fragment is 300bp, and the four bases (A/C/T/G) of the 1 st to 60 th bp, 61 st to 120 th bp, 121 st to 180 th bp, 181 th to 240 th bp, 241 th to 300 th bp of each subsection and the whole 1 st to 300 th bp of each subsection account for 25% respectively. Meanwhile, the sequence insert and the standard substance for sequencing library quantification obtained by amplification thereof cannot be compared with human beings, common animals and plants and microorganisms, so that the pollution of the related sequence of the standard substance for sequencing library quantification possibly caused by various reasons can be effectively avoided, and the accuracy of sequencing data result analysis of a sample of a library to be detected is improved.
The length of the standard for sequencing library quantification will vary from sequencing platform to sequencing platform with different linker lengths. The invention relates to a standard composition structure for quantifying a high throughput sequencing library, which comprises but is not limited to the following structural sequences:
the illuminea sequencing platform sequences the library quantitative standard structural sequence:
5'-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCTT
sequence insert (SEQ ID NO. 1)
-AAGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCGTCTTCTGCTTG-3'。
The Huada sequencing platform sequences the library quantitative standard substance structural sequence:
5'-CTCTCAGTACGTCAGCAGTTCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTT
sequence insert (SEQ ID NO. 1)
-AAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAACTGATAAGGTCGCCATGC-3'。
The base ratio of the standard substance for quantitative sequencing library in the invention is balanced in a plurality of section ranges, and when the standard substance for quantitative sequencing library is used for real-time fluorescence quantitative PCR, the amplification efficiency is high and stable, and the method is beneficial to avoiding poor or unstable amplification efficiency caused by complex sequence of the standard substance for quantitative sequencing library.
Preferably, the copy concentration of the target sequence after PCR amplification is not less than 0.5nM.
Preferably, after PCR amplification, quantitative determination of the library of specific sequences of interest is performed using digital PCR and the copy concentration of the sequences of interest is diluted in a gradient.
In a more specific embodiment provided by the invention, a set of library quantification standards are obtained by dilution: quantitative determination of the library of the specific sequences of interest was performed using digital PCR and the copy concentration was converted to molar concentration. Library quantification was diluted with standard to a range of concentration gradients (e.g., 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005 nM) and poly (dN) (N=A/C/T/G bases) was added during formulation. Poly (dN) is preferably Poly (dA) with a concentration of 0.1-100 ng/. Mu.L, preferably 1-10 ng/. Mu.L, preferably 1 ng/. Mu.L; the number of dA bases is 5 to 100, preferably 10 to 50, preferably 18 to 30.
In a second aspect, the invention provides a preparation method of the standard substance for quantifying the high-throughput sequencing library, which is obtained by PCR amplification of template DNA comprising a sequence insert, wherein the nucleotide sequence of the sequence insert is shown as SEQ ID NO. 1.
Preferably, the nucleotide sequence of the upstream primer of the primer pair for PCR amplification is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.
The preparation method provided by the invention is simple, rapid, stable and reliable. The obtained high-throughput sequencing library has single sequence of standard substance, no need of library fragment screening and small batch-to-batch difference; and has high stability, and is convenient for use and storage.
In a third aspect, the invention provides an application of the standard for high-throughput sequencing library quantification or the standard for high-throughput sequencing library quantification prepared by the preparation method in library quantification.
Preferably, the application of the invention carries out real-time fluorescence quantitative PCR detection on the standard substance for high-throughput sequencing library quantification and the library sample to be detected together, draws a standard curve according to the molar concentration and Ct value of the standard substance for high-throughput sequencing library quantification, and calculates the molar concentration of the library sample to be detected according to the standard curve and the Ct value of the library sample to be detected.
Preferably, in the application of the invention, the amplification efficiency of the standard curve is 95-105%, and the linear correlation coefficient r is more than 0.999.
The beneficial effects are that:
the invention provides a standard substance for quantitative use of a high-throughput sequencing library, a preparation method and application thereof. The high-throughput standard substance for sequencing library quantification is obtained by PCR amplification of template DNA comprising sequence insert fragments, and the sequence insert fragments and the standard substance for sequencing library quantification obtained by amplification thereof cannot be compared with human beings, common animals, plants and microorganisms, so that the pollution of related sequences of the standard substance for sequencing library quantification possibly caused by various reasons can be effectively avoided, and the accuracy of sequencing data result analysis of a sample of a library to be detected is improved. Meanwhile, the base ratio of the standard substance for quantitative sequencing library provided by the invention in the range of a plurality of sections is balanced, and when the standard substance for quantitative sequencing library is used for real-time fluorescence quantitative PCR, the amplification efficiency is high and stable, and the phenomenon that the amplification efficiency is poor or unstable due to the complex sequence of the standard substance for quantitative sequencing library is avoided.
Detailed Description
The invention provides a standard substance for quantitative use of a high-throughput sequencing library, a preparation method and application thereof. The standard for high-throughput sequencing library quantification is obtained by PCR amplification of template DNA comprising sequence inserts.
In one specific embodiment provided by the invention, the preparation and application method of the standard for quantifying the high-throughput sequencing library comprises the following steps:
(1) 1 specific sequence library plasmids were synthesized: the library of specific sequences on the plasmid includes a double-ended linker and a specific sequence insert (SEQ ID NO. 1). Wherein the length of the insert of the specific sequence is 300bp (the length of the standard substance for quantitative analysis of the sequencing library is different due to the different lengths of the joints of different sequencing platforms), and the four bases (A/C/T/G) of each subsection of 1 to 60bp, 61 to 120bp, 121 to 180bp, 181 to 240bp, 241 to 300bp and the whole section of 1 to 300bp of the insert of the specific sequence are 25% respectively. Meanwhile, the insert fragment of the specific sequence can not be compared with human, common animals, plants and microorganisms, so that data pollution can be effectively prevented.
(2) 1 library of specific sequences of interest was obtained: 1 library of specific sequences of interest on the plasmid was obtained using the PCR method. The library of specific sequences of interest comprises a double-ended linker and a specific sequence insert (SEQ ID NO. 1). The sequence of the target specific sequence library is single and fixed, and the ratio of four bases (A/C/T/G) of each subsection of 1 to 60bp, 61 to 120bp, 121 to 180bp, 181 to 240bp, 241 to 300bp of the specific sequence and the whole subsection of 1 to 300bp is 25 percent. The target specific sequence library needs to be close to the length of the sample of the library to be detected (the length is up to +/-100 bp), and the excessive difference is avoided, so that the target specific sequence library and the sample of the library to be detected can carry out non-differential amplification reaction when the library is quantified.
(3) Dilution to obtain a set of library quantification standards: quantitative determination of the library of the specific sequences of interest was performed using digital PCR and the copy concentration was converted to molar concentration. Library quantification was diluted with standard to a range of concentration gradients (e.g., 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005 nM) and poly (dN) (N=A/C/T/G bases) was added during formulation. Poly (dN) is preferably Poly (dA), and the concentration of the Poly (dA) is 0.1-100 ng/. Mu.L, preferably 1-10 ng/. Mu.L, preferably 1 ng/. Mu.L; the number of dA bases is 5 to 100, preferably 10 to 50, preferably 18 to 30.
The preparation method of the standard substance for quantitative high-throughput sequencing library provided by the invention can be used for rapidly, stably and accurately preparing the standard substance for quantitative high-throughput sequencing library with specific sequence and length, and effectively reducing the risk caused by inaccurate quantitative sequencing library to be detected.
The invention applies the obtained standard for quantitative high-throughput sequencing library to library quantification: and carrying out real-time fluorescent quantitative PCR detection on the high-throughput sequencing library quantitative standard substance and a library sample to be detected. After qPCR data is taken off the machine, a standard curve is firstly prepared by using a group of standard substance results for sequencing library quantification, wherein the X axis and the Y axis are respectively the logarithmic value of the molar concentration of the standard substance for sequencing library quantification and the Ct value of the standard substance for sequencing library quantification, and a standard curve formula (Y=kX+b, wherein k and b are respectively the slope and intercept of the standard curve) is obtained through straight line fitting. And substituting the Ct value of the library sample to be detected into a linear standard curve, and obtaining the molar concentration (the unit is consistent with that of the library standard substance) of the library sample to be detected. And (5) loading the quantified library sample to be detected according to the requirement of high-throughput sequencing.
The standard substance sequence for sequencing library quantification is single in length and fixed, and meanwhile, balanced bases in a plurality of section ranges are beneficial to improving the amplification efficiency and stability of real-time fluorescence quantitative PCR, and the accuracy and stability of the quantitative result of a library sample to be detected are guaranteed, so that relatively accurate high-quality sequencing data are rapidly and stably obtained, and the problem that the amplification efficiency is poor or unstable due to the fact that the sequence of the standard substance for sequencing library quantification is complex is effectively avoided.
Since the adaptors of sequencing libraries corresponding to different sequencing platforms may be different, embodiments are described below using the illuminea sequencing platform as an example.
The preparation method mainly comprises the following steps:
s1, synthesizing 1 escherichia coli cloning plasmid (or long-chain DNA molecule) containing the insertion fragment of the specific sequence by adopting a gene synthesis method.
Embodiments are described with respect to plasmids: plasmid vectors are cloning plasmids, including but not limited to pUC57 plasmids. Inserts on plasmids included the double-ended linker of the sequencing library and the insert of the specific sequence (SEQ ID NO. 1). The specific sequence insertion fragment can not be aligned with human, common animals, plants and microorganisms, and can effectively prevent data pollution. The correct synthesis of the plasmid insert sequence was confirmed by Sanger sequencing.
S2, extracting and purifying the plasmid by adopting a conventional molecular biology method (for example, using a commercial kit), and detecting the mass concentration of the plasmid (for example, using a spectrophotometer, a fluorometer and the like).
S3、PCR
(1) Taking out the plasmid sample of the specific sequence library for preparing the standard substance for sequencing library quantification and the reagents in the table below, thawing at room temperature, gently oscillating and uniformly mixing after complete thawing, and placing the mixture on an ice box for standby after short centrifugation.
(2) The PCR reaction system was prepared in a centrifuge tube of 0.2. 0.2 mL according to the following table. Wherein the plasmid template dosage is as follows: 1pg-50ng, preferably 1pg-1ng, preferably 10pg. High fidelity DNA polymerase is used to ensure fidelity of the DNA sequence during amplification.
TABLE 1 PCR reaction System
The specific primer sequences are shown below:
forward primer (P5): AATGATACGGCGACCACCGAG
Reverse primer (P7): CAAGCAGAAGACGGCATACGAGA
(3) After the prepared reaction system was subjected to shaking and centrifugation, PCR was performed under the following reaction conditions, and the temperature was 105 ℃.
TABLE 2 PCR reaction conditions
Note that: the smaller the amount of plasmid template corresponding to the specific sequence library, the more cycles should be.
S4, magnetic bead purification after PCR reaction
After the PCR reaction is completed, the sample is transferred to an operation table for the next purification operation. According to the product instruction, VAHTS is used ® DNA Clean Beads purification magnetic Beads purification PCR products.
S5, preparing standard substance for sequencing library quantification
(1) 1 [ mu ] L of a specific sequence library is taken for mass concentration detection (for example, a spectrophotometer, a fluorometer and the like) and corresponding molar concentration is calculated roughly according to the following formula.
Molar concentration (nM) =mass concentration (ng/. Mu.L)/(molecular weight (g/mol)) 10-6
(2) Based on the roughly calculated molar concentration of the library of specific sequences, it is diluted to the concentration range required for digital PCR detection.
(3) Taking out the reagents in the table below, thawing at room temperature, gently oscillating and uniformly mixing after complete thawing, and placing the mixture on an ice box for standby after short centrifugation.
(4) In a centrifuge tube of 0.2. 0.2 mL, digital PCR reaction systems were prepared according to the following tables, respectively.
TABLE 3 digital PCR reaction System
In a digital PCR reaction system:
PCR forward primer (P5): AATGATACGGCGACCACCGAG;
PCR reverse primer (P7): CAAGCAGAAGACGGCATACGAGA.
(5) And (3) carrying out shaking mixing centrifugation on the prepared digital PCR reaction system, and carrying out digital PCR reaction according to the following reaction conditions.
TABLE 4 PCR reaction conditions
(6) Copy concentration obtained by digital PCR was converted to molar concentration. Molar concentration (mol/L) =copy concentration (copy/L)/(avogaldel constant).
(7) The specific sequence library is diluted according to 10 times gradient concentration, and poly (dN) with a certain concentration is added at the same time, so that a group of standards (such as 0.5nM, 0.05nM, 0.005nM, 0.0005nM and 0.00005 nM) for quantitative sequencing library are prepared. Poly (dN) is preferably Poly (dA) with a concentration of 0.1-100 ng/. Mu.L, preferably 1-10 ng/. Mu.L, preferably 1 ng/. Mu.L; the number of dA bases is 5 to 100, preferably 10 to 50, preferably 18 to 30.
S6, carrying out real-time fluorescence quantitative PCR detection on the standard substance for sequencing library quantification and the library sample to be detected. After qPCR data is taken off the machine, a standard curve is firstly prepared by using a group of standard substance results for sequencing library quantification, wherein the X axis and the Y axis are respectively the logarithmic value of the molar concentration of the standard substance for sequencing library quantification and the Ct value of the standard substance for sequencing library quantification, and a standard curve formula (Y=kX+b, wherein k and b are respectively the slope and intercept of the standard curve) is obtained through straight line fitting. And substituting the Ct value of the library sample to be detected into a linear standard curve, and obtaining the molar concentration (the unit is consistent with that of the library standard substance) of the library sample to be detected.
S7, loading the quantified library sample to be detected according to the requirement of high-throughput sequencing.
S8, aiming at the data after the machine is started, the known sequence of the standard substance for quantifying the sequencing library can be removed from the sequencing result through sequence comparison, so that the analysis of the sequencing result of the sample of the library to be detected is prevented from being influenced.
In order to facilitate understanding of the present invention, the following description will be given for clarity and completeness of the technical solutions in the embodiments of the present invention, but they should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents and equipment used were conventional products available commercially without the manufacturer's attention.
Example 1
In this example, using the illuminea sequencing platform as an example, a standard for quantitative determination of a test library was prepared according to the description of the specific embodiment, and 3 days were selected for batch preparation, and one batch was prepared each day. The standard substance for quantitative analysis of the test library is designed into a group of 5 standard substances, and the concentration is as follows: 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM. Poly (dN) is selected from Poly (dA), and the concentration of the Poly (dA) is 1 ng/. Mu.L; the number of dA bases is 18.
3 prepared standard substances for sequencing library quantification in batches are subjected to repeated quantification (the quantifying reagent is KAPA Library Quantification Kit) for 3 times on 10 samples of the library to be tested in the same group, and after each quantification, the sequencing is performed on the machine according to the measured concentration of the 10 samples of the library to be tested. And simultaneously setting a control group: namely, 10 samples of the library to be detected are quantified by using a Qubit fluorometer, and after quantification, on-machine sequencing (sequencer model number: illumina Nextseq CN500,500) is carried out according to the actual measured Qubit concentration of the 10 samples of the library to be detected, wherein the expected data volume of each sample is 4G.
The result shows that the relative standard deviation among the concentration of 10 to-be-detected libraries quantified by using a sequencing library quantification standard substance obtained by a real-time fluorescence quantitative PCR method (qPCR) is not more than 10%; the relative standard deviation among the library concentrations of 10 samples to be detected, which are quantified by using a standard substance for quantifying the sequencing library obtained by a control group (Qubit quantification) method, is 22% at maximum. It can thus be shown that: the sequencing library prepared by the method has small batch-to-batch variation when quantitatively prepared by using the standard substance. The specific results are shown in the following table.
TABLE 5 quantitative results of library from different methods
And (3) loading the 10 quantitative library samples to be detected according to the high-throughput sequencing requirement. Sequencing was performed by a high throughput sequencer, exemplified by a illumina Nextseq CN500 sequencer. The results show that: the relative deviation between the actual output data amount and the expected data amount of three batches of 10 to-be-detected libraries quantified by using a standard substance for sequencing library quantification obtained by a real-time fluorescence quantitative PCR method (qPCR) is not more than +/-10%. And the relative deviation of the actual output data quantity and the expected data quantity of 10 sample libraries to be detected, which are quantified by using a standard substance for quantifying the sequencing library obtained by a control group (Qubit quantification, only last machine) method, is maximally up to-30%. It can thus be shown that: the sequencing library prepared by the method has small batch-to-batch variation when quantitatively prepared by the standard substance, and the actual output of the library sequencing data quantity quantitatively prepared by the method is more consistent with the expected output. The specific results are shown in the following table.
TABLE 6 quantitative comparison of sequencing data from libraries by different methods
Example 2
The library quantification standard is prepared according to the specific embodiment, and the library quantification standard is designed into a group of 5 standards, and the concentration is as follows: 0.5nM, 0.05nM, 0.005nM, 0.0005nM, 0.00005nM. Poly (dN) is Poly (dA), and the concentration of Poly (dA) is 1 ng/. Mu.L; the number of dA bases is 18. A control group was also set, and the control group was not added with poly (dN) as described in the embodiment. The two groups of libraries were tested for real-time stability (stored at-30 to-10 ℃ C., 0/4/8/12/16/20/24 months) and accelerated stability to failure (repeated freeze thawing 10 times, standing at room temperature (20-30 ℃ C.) for 2 days) simultaneously with standard substances. And (3) testing the standard substances for library quantification in total, and respectively quantifying the library samples to be tested in the same instance.
The quantitative result shows that the standard substance for quantitative analysis of the prepared test library has the amplification efficiency of the standard curve between 95 and 105 percent, the linear correlation coefficient r is larger than 0.999, and no obvious difference exists between groups no matter the quantitative result is real-time stability test or accelerated damage stability; in the control group (without adding poly (dN) described in the specific embodiment), the amplification efficiency of the standard curve obtained by calculation is partially between 90 and 95 percent, the linear correlation coefficient r is partially between 0.98 and 0.99, and the quantitative result of the same sample to be detected library sample is greatly fluctuated no matter the real-time stability test or the accelerated damage stability test is carried out, the time is prolonged, the accelerated damage condition is increased. Therefore, the sequencing library prepared by the method has good real-time stability and accelerated damage stability (repeated freeze thawing and room temperature placement) when being prepared by the standard substance quantitatively. The specific results are shown in tables 7 to 10 below.
TABLE 7 real-time stability analysis results (PCR amplification efficiency and Linear correlation coefficient) of standards for library quantification
Table 8 results of real-time stability analysis of standards for quantitative analysis of library (quantitative concentration results)
TABLE 9 analysis of stability to accelerated disruption of library quantification Using standards (PCR amplification efficiency and Linear correlation coefficient)
TABLE 10 analysis results of accelerated stability to failure of standards for quantification of library (concentration quantification results)
The above examples merely represent several embodiments of the present invention, which facilitate specific and detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. The standard substance for high-throughput sequencing library quantification is characterized in that the standard substance is obtained by PCR amplification of template DNA comprising a sequence insert, and the nucleotide sequence of the sequence insert is shown as SEQ ID NO. 1.
2. The high throughput sequencing library quantitative standard according to claim 1, wherein the copy concentration of the target sequence after PCR amplification is not less than 0.5nM.
3. The standard for quantitative determination of high throughput sequencing library according to claim 2, wherein after PCR amplification, quantitative determination of the library of target specific sequences is performed using digital PCR and the copy concentration of target sequences is subjected to gradient dilution.
4. The high throughput sequencing library quantitation standard of claim 3, wherein the high throughput sequencing library quantitation standard further comprises poly (dN), n=a/C/T/G bases.
5. The high throughput sequencing library quantification standard of claim 4, wherein the poly (dN) is poly (dA) at a concentration of 0.1-100 ng/. Mu.L and a dA base number of 5-100.
6. The method for preparing the standard substance for quantifying the high throughput sequencing library according to any one of claims 1 to 5, wherein the standard substance is obtained by PCR amplification of a template DNA comprising a sequence insert, and the nucleotide sequence of the sequence insert is shown as SEQ ID NO. 1.
7. The method of claim 6, wherein the nucleotide sequence of the upstream primer of the primer pair for PCR amplification is shown in SEQ ID NO.2 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 3.
8. Use of a standard for high throughput sequencing library quantification according to any one of claims 1 to 5 or a standard for high throughput sequencing library quantification according to any one of claims 6 to 7 in library quantification.
9. The use according to claim 8, wherein the high throughput sequencing library quantitative standard is subjected to real-time fluorescent quantitative PCR detection together with the library sample to be detected, a standard curve is drawn according to the molar concentration and Ct value of the high throughput sequencing library quantitative standard, and the molar concentration of the library sample to be detected is calculated according to the standard curve and Ct value of the library sample to be detected.
10. The use according to claim 9, wherein the standard curve has an amplification efficiency of 95-105% and a linear correlation coefficient r > 0.999.
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