CN107354207B

CN107354207B - liquid phase hybridization capture kit based on double-stranded probe, washing kit and application thereof

Info

Publication number: CN107354207B
Application number: CN201710560804.0A
Authority: CN
Inventors: 赵金银; 李宏志; 姜旭; 刘琦; 许立志; 于闯; 李�杰
Original assignee: Dalian Gentalker Biotechnology Co Ltd
Current assignee: SHUANGDI, INC.
Priority date: 2017-07-11
Filing date: 2017-07-11
Publication date: 2019-12-10
Anticipated expiration: 2037-07-11
Also published as: CN107354207A

Abstract

The invention discloses a genome sequencing library hybridization capture kit, a washing kit and application of the kit and the washing kit in liquid phase hybridization capture. The capture kit comprises a 2 × concentration solution and 100% deionized formamide, wherein the 2 × concentration solution comprises 100-150mM sodium dihydrogen phosphate, 4 × sodium citrate buffer solution, 1-3mM ethylene diamine tetraacetic acid, 2 × Denhardt solution, 2-5mg/ml Carrier RNA and 15-20 w/w% dextran sulfate, and the 4 × sodium citrate solution comprises 0.6M sodium chloride, 0.06M sodium citrate and pH 7.0; and the 2 xdenhardt solution comprises 0.04% ficoll 400, 0.04% polyvinylpyrrolidone, and 0.04% bovine serum albumin.

Description

Liquid phase hybridization capture kit based on double-stranded probe, washing kit and application thereof

Technical Field

The invention belongs to the field of molecular biology, and relates to a genome sequencing library hybrid capture kit, a washing kit and application thereof in liquid phase hybrid capture.

Background

The emergence of a New Generation Sequencing (NGS) technology in the beginning of the 20 th century brought about a rapid progress in the field of DNA Sequencing technology. Compared with the first generation sequencing technology, the second generation sequencing technology has the technical characteristics of high throughput and high accuracy, and can simultaneously sequence hundreds of millions of DNA fragments. Compared with the traditional Sanger method for sequencing, the cost of NGS sequencing is greatly reduced. However, the whole genome sequencing cost is still high, and many research applications do not require whole genome sequencing, and the requirement for sequencing target regions of one or more samples is urgent, so that the targeted sequencing method for the target regions can significantly reduce the sequencing cost, and can also shorten the sequencing time and the biological information analysis period. The advent of target sequence capture technology has satisfied the above needs. The target sequence capture sequencing is to design a probe for a known specific genome region and then perform sequencing, wherein DNA fragments which are not interested in the target enrichment process are removed to the maximum extent, so that the result generated by sequencing is mainly focused on the target region of interest. Currently, the commonly used sequence capture methods are: PCR, MolecuLar Inversion Probe (MIP), and hybrid capture. The PCR method has the advantages of high sensitivity, high specificity, good repeatability and the like, but the method cannot easily expand a target region, is suitable for capturing a few small regions, is difficult to amplify part of the regions, and has no good solution to the problems of low somatic mutation detection sensitivity and the like. The molecular inversion probe method has the advantages of simple sample pretreatment, small sample demand and the like, but the molecular probe has higher synthesis cost and poorer uniformity and is difficult to capture genome sequences with larger regions. The hybridization capture method captures and separates a target sequence by a specific hybridization mode of a target DNA fragment and a probe which is already provided with a biotin label. The technology is widely applied to a second-generation sequencing platform, and particularly, the liquid phase hybridization capture method has unique advantages in the technical field of target region capture. The liquid phase hybridization capture technology can overcome the problems of the PCR method and the molecular inversion probe method, and has the advantages of better capture efficiency, less preference, higher detection sensitivity, simpler operation, proper cost and the like.

The high capture efficiency and accuracy of the liquid phase hybridization capture technology mainly depend on three aspects: 1. the accuracy and specificity of the probe; 2. hybridization buffer solution environment and hybridization conditions; 3. washing buffer environment and washing conditions after hybridization. The high accuracy and specificity of the biotinylated probes is guaranteed by the high hybridization accuracy, which is the level that the existing probe synthesis techniques in the art can achieve, e.g., IDTa probe synthesis technique. The finding of a high-efficiency hybridization buffer solution, hybridization conditions, a washing buffer solution and a washing method is particularly important, and the link is a main bottleneck and speed-limiting step for effectively improving the hybridization efficiency, improving the capture accuracy, simplifying the experimental process and reducing the cost.

Therefore, based on the research results and technical characteristics, the development of a high-efficiency, high-accuracy and low-cost liquid phase hybridization capture reagent and a method thereof is urgent to meet the increasing requirements on targeted sequencing.

Disclosure of Invention

The present invention relates to novel compositions, related kits and hybridization washing methods for hybridization and washing of double-stranded nucleic acids. These compositions and methods enable faster, more efficient, and better capture of targeted regions of genomic DNA enrichment than traditional processes.

One aspect of the present invention provides a genomic sequencing library hybrid capture kit comprising a 2 × concentration solution and 100% deionized formamide,

Wherein the 2 Xconcentration solution comprises 100-150mM sodium dihydrogen phosphate, 4 Xsodium citrate buffer solution, 1-3mM ethylene diamine tetraacetic acid, 2 XDenhardt solution, 2-5mg/ml Carrier RNA and 15-20 w/w% dextran sulfate,

Wherein the 4 x sodium citrate solution comprises 0.6M sodium chloride, 0.06M sodium citrate, ph 7.0; and is

The 2 xdenhardt solution contained 0.04% ficoll 400, 0.04% polyvinylpyrrolidone, and 0.04% bovine serum albumin.

Preferably, the 4 × sodium citrate solution is prepared by diluting a 20 × sodium citrate solution by 5 times, wherein the 20 × sodium citrate solution comprises 3M sodium chloride, 0.3M sodium citrate, and has a ph of 7.0; and is

the 2 XDenhardt solution was prepared by 25-fold dilution of 50 XDenhardt solution, which contained 1% ficoll 400, 1% polyvinylpyrrolidone, and 1% bovine serum albumin.

In a preferred embodiment, the 2 Xconcentration solution comprises 100mM sodium dihydrogen phosphate, 4 Xsodium citrate buffer, 2mM ethylenediaminetetraacetic acid, 2 XDenhardt's solution, 2mg/ml Carrier RNA and 20 w/w% dextran sulfate.

In another aspect of the present invention, a method for using the genomic sequencing library hybrid capture kit is provided, which comprises the following steps:

Mixing the 2 × concentration solution with the 100% deionized formamide and probe solution according to the following conditions:

the 2 × concentration solution is diluted by one time in the final solution, and

the concentration of the deionized formamide in the final solution is 20-50 v/v%.

In yet another aspect, the present invention provides a genomic sequencing library hybrid capture solution obtained by the above-described method of use.

In yet another aspect, the present invention provides a wash solution kit for use after hybrid capture of a genomic sequencing library, comprising the following 5 wash buffer components:

1) Stringent wash solution: 2 × sodium citrate buffer, 30% v/v% deionized formamide;

2) Washing solution I: 2 × sodium citrate solution, 0.1% Sodium Dodecyl Sulfate (SDS);

3) Washing solution II: 2 x sodium citrate solution;

4) Washing solution III: 0.2 x sodium citrate solution; and

5) Streptavidin magnetic bead washing solution: 10mM Tris-HCl pH 7.5-8.0, 1mM EDTA and 2M NaCl,

Wherein the 2 x sodium citrate solution comprises 0.3M sodium chloride and 0.03M sodium citrate, pH7.0, and

The 0.2 x sodium citrate solution contained 0.03M sodium chloride and 0.003M sodium citrate, pH 7.0.

Preferably, the 2 x sodium citrate solution and the 0.2 x sodium citrate solution are each dilutions of a 20 x sodium citrate solution, the 20 x sodium citrate solution comprising 3M sodium chloride and 0.3M sodium citrate, pH 7.0.

in another aspect of the present invention, a genomic sequencing library hybridization kit is provided, which comprises the above hybridization capture kit and the above washing solution kit.

In another aspect of the invention, the genome sequencing library hybridization kit is used in hybridization capture enrichment of a genome DNA sequencing library by a probe.

In a further aspect, the present invention provides a liquid phase hybrid capture method wherein the above hybrid capture kit and/or the above wash solution kit are used in said method.

Advantageous effects

the double-stranded nucleic acid liquid phase hybridization capture solution and the washing solution kit provided by the invention have higher efficiency, and can shorten the hybridization time to 16h, while the general hybridization time of a commercial kit is 64-72h, so that the test period can be greatly shortened, and the cost is reduced; the washing solution has a good elution effect, greatly reduces DNA fragments in non-target areas, effectively improves the targeted enrichment efficiency and reduces the cost.

Drawings

FIG. 1 shows the results of comparison of the fold enrichment of 4 target genes in three experimental groups.

FIG. 2 shows the results of comparison of the fold enrichment of 2 non-target genes in the three experimental groups.

the experiment group 1 uses a commercial kit hybridization solution and a washing solution, the experiment group 2 and the experiment group 3 use the capture solution and the washing solution kit, the experiment conditions are the same, and the performance of the self-made reagent is evaluated. The enrichment fold of the target gene and the non-target gene was detected by real-time quantitative PCR (realtime-qPCR). The results were the mean. + -. standard error (mean. + -. SEM) of three independent replicates.

Detailed Description

The term "2 × concentration solution" in the present invention means that the concentration of the solution is 2 times that of the working solution at the time of use, and is also understood to mean that the solution is diluted one time at the time of use.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that many technical details are set forth in the embodiments of the present invention in order to provide a better understanding of the present invention to the reader. However, the technical solutions claimed in the claims of the present application can be implemented even without these technical details.

Denhardt's solvent used in the following examples of the invention was purchased from Amresco, Carrier RNA from Tiangen Biotechnology, Inc., and deionized formamide liquid from Solebao Biotechnology, Inc.

examples

Preparative example 1 preparation of Capture solution

The solution is prepared from sodium dihydrogen phosphate (NaH)₂PO₄) Buffer solution, sodium citrate buffer solution (SSC), and ethylenediaminetetraacetic acid

(EDTA), deionized formamide, Denhardt (Denhardt) solution, Carrier RNA and dextran sulfate

and (3) preparing. First, 2 Xconcentration solution (without deionized formamide, deionized formamide liquid is added separately

Obtained in solution), can be preserved for a long time at the temperature of 20 ℃, and has the following specific formula:

100mM NaH₂PO₄

4×SSC

2mM EDTA

2 XDenhard solution

2mg/ml Carrier RNA

20 w/w% dextran sulfate

pH of the solution was 7.0

The capture solution comprises a 2 x concentration solution mixed with 100% formamide liquid to form a hybrid capture solution working solution, for example, in the volumetric mixing ratios shown in table 1 below.

TABLE 1

Preparation example 2 preparation of washing solution kit

The washing solution kit comprises 5 washing buffer solution components, can be stored for a long time at the temperature of-20 ℃, and has the following specific formula:

Stringent wash solution: 2 XSSC solution, 30 v/v% deionized formamide.

washing solution I: 2 XSSC solution, 0.1% Sodium Dodecyl Sulfate (SDS).

Washing solution II: 2 XSSC solution.

Washing solution III: 0.2 XSSC solution.

biotin affinity magnetic bead washing solution: 10mM Tris-HCl, pH 7.5; 1mM EDTA; 2M NaCl.

example 1 use of Capture solution and Wash solution kits in hybridization Capture enrichment

Specifically, the whole experimental flow comprises: firstly, breaking the extracted sample DNA into DNA fragments of 200-250bp by ultrasonic (but not limited to an ultrasonic breaking method), and connecting the DNA fragments with index joints through end repairing, A adding and connecting processes; and then hybridizing the constructed capture library with DNA fragments containing the target gene region to be captured, capturing the DNA fragments of the target region, evaluating the enrichment effect of the DNA fragments of the target region, and sequencing the DNA fragments. And obtaining a sample detection result after bioinformatics data analysis.

1. Sample genomic DNA extraction and library preparation

Method for extracting genomic DNA of sample A human whole Blood genomic sample designated as S1 was extracted with reference to QIAamp DNA Blood Mini Kit (Qiagen). Fragmenting the obtained high-quality genome DNA sample into 200-250bp DNA fragments, dividing the sample into 3 parts, performing a parallel library construction experiment, then respectively connecting 3 groups of DNA fragments with 3 different index joints (related reagents are from KAPA Hyper PrepKi Illumina platforms) through processes of end repair, A addition, connection and the like, and obtaining a sample DNA library after PCR amplification and purification. The index sequences used for the three samples are as follows: s1-1: ATCACG; s1-2: CGATGT; s1-3: TTAGGC.

2. genomic library mixing, blocking and drying

The constructed genomic DNA libraries were mixed according to the data amount distribution ratio to a total amount of 1ug, and then the COT DNA, the genomic DNA libraries and the blocking primers were mixed according to the ratio as in Table 2 below. The COT DNA is used as a part of DNA fragments with high repetition rate in a genome, the hybridization efficiency is improved during hybridization, and the blocking primer is used for blocking a sequencing joint in a library. The mixed sample is evaporated to dryness in a vacuum concentrator at 60 ℃ for subsequent hybridization.

TABLE 2

3. Re-solubilization and denaturation: to the above dried mixture was added 10.5. mu.L of the capturing solution. Experiment group 1, sample number S1-1, using Roche NimbleGen commercial kit hybridization reagent; experimental group 2, sample No. S1-2, using the capture solution prepared as described in preparation example 1 above; experiment group 3, sample number S1-3, is a repeat of experiment group 2. After being fully dissolved by vortex, the mixture is denatured at 95 ℃ for 10 min.

4. And (3) hybridization: the denatured 10.5. mu.L of the mixture was added to a library of 4.5. mu.L hybrid capture probes, and three experiments performed liquid phase hybrid capture using the same double-stranded DNA probe, vortexed for 30s, fully solubilized and mixed, and centrifuged at full speed for 30 s. Experiment 1 was hybridized at 47 ℃ for 64h, and experiment 2, 3 were hybridized at 55 ℃ for 16 h.

5. streptavidin magnetic beads capture target region DNA library fragments: after hybridization reaction, 15 μ L of sample was transferred to 100 μ L of streptavidin-labeled magnetic beads previously resuspended by washing with Bead Wash Buffer (panel 1) in Roche NimbleGen commercial kit and streptavidin magnetic Bead washing solution (panels 2 and 3) prepared in preparation example 2, respectively, after mixing, panel 1 was incubated at 47 ℃ for 45min, panels 2 and 3 were incubated at 55 ℃ for 45min, mixing was performed every 15 min, captured fragments were bound to magnetic beads, target fragments were specifically adsorbed, and hybridized fragments were captured.

Experiment group 1, sample number S1-1, elution reagent using Roche NimbleGen commercial kit; experimental group 2, sample No. S1-2, using the washing solution prepared as described in preparation example 2 above; experiment group 3, sample number S1-3, is a repeat of experiment group 2.

6. washing non-target region DNA fragments: here, the elution step of the elution reagent prepared in the above preparation example 2 is mainly described.

The experimental group 1 was added with 100. mu.L of washing solution I of Roche NimbleGen commercial kit preheated at 47 ℃ and the experimental groups 2 and 3 were added with 100. mu.L of washing solution I prepared in the above preparation example 2 preheated at 55 ℃, and after mixing, the mixture was magnetically suspended and the supernatant was discarded, and the procedure was repeated once.

② adding 200 μ L of Stringent Wash Buffer preheated at 47 ℃ into the experimental group 1, adding 200 μ L of Stringent washing solution preheated at 55 ℃ prepared in the preparation example 2 into the experimental groups 2 and 3, uniformly mixing, incubating at 55 ℃ for 5min, magnetically suspending, discarding the supernatant, repeating for three times and four times.

③ the washing solution I of the elution reagent of the Roche NimbleGen commercial kit is added into the experimental group 1, and 200. mu.L of the washing solution I prepared in the preparation example 2 is added into the experimental groups 2 and 3, vortexed for 2min, and the supernatant is discarded by magnetic suspension.

Adding the washing solution II of the elution reagent of the Roche NimbleGen commercial kit into the experimental group 1, adding 200 mu L of the washing solution II prepared in the preparation example 2 into the experimental groups 2 and 3, vortexing for 1min, and removing the supernatant through magnetic suspension.

Fifthly, adding the washing solution III of the elution reagent of the Roche NimbleGen commercial kit into the experimental group 1, adding 200 mu L of the washing solution III prepared in the preparation example 2 into the experimental groups 2 and 3, whirling for 30s, and magnetically suspending and discarding the supernatant.

sixthly, adding 50 mu L of PCR level dd H₂O resuspend the magnetic beads.

7. Enrichment after capture: after elution, the magnetic beads carry the captured target DNA fragments and enter LM-PCR enrichment of the sample. The LM-PCR reaction is shown in Table 3 below (one capture sample for two-tube PCR reaction).

TABLE 3

The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 45 s; denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 14 cycles; extending for 1min at 72 ℃; keeping the temperature at 4 ℃.

After reaction, purifying PCR amplification products by using purified magnetic beads AMPure beads to obtain a captured sample library, and performing quality inspection and sequencing analysis.

experimental example 1 enrichment factor determination of target region and non-target region by fluorescent quantitative PCR

And measuring the enrichment multiples of the target region and the non-target region before and after enrichment by using an SYBR Green method. The reaction system is shown in table 4 below.

TABLE 4

The PCR reaction conditions are as follows: 10min at 95 ℃; at 95 ℃ for 10s and 60 ℃ for 1min for 40 cycles; 95 ℃ for 10s and 65 ℃ for 1 min. And measuring the Ct value of the sample through the acquired signal, wherein the measured Ct value of the sample depends on the initial concentration of the template DNA in the reaction, and the lower the Ct value is, the higher the initial concentration of the template DNA is, so that the enrichment times of the sample can be evaluated according to the difference change of the Ct values of the sample before and after enrichment. The enrichment of the target region and the non-target region in the three experimental groups based on the above experiments is shown in the figure. FIG. 1 shows that the enrichment times of 4 target regions in the experimental groups 2 and 3 are obviously higher than that of 4 target regions in the experimental group 1, so that the hybridization capture efficiency of the self-made reagent is obviously higher than that of the commercialized kit. Fig. 2 shows that the enrichment times of 2 non-target regions in experimental groups 2 and 3 are obviously lower than that of 2 non-target regions in experimental group 1, so that the elution efficiency of the self-made reagent is obviously higher than that of the commercial kit, and background non-target fragments can be effectively removed.

Experimental example 2 high throughput sequencing and data analysis

The method comprises the steps of performing high-throughput sequencing by adopting a second-generation sequencing platform, performing sequencing on a captured sequence on an Illumina NextSeq 500 sequencing platform (sequencing reagents are purchased from Illumina commercial sequencing kit), analyzing obtained data to obtain a tag sequence on the captured sequence, determining a corresponding sample DNA source according to the tag sequence, and comparing according to a target region to obtain effective data of the target region so as to obtain target region information of the sample. The sequencing library obtained by the above method was sequenced using a second generation sequencing approach and sequencing data were obtained, and the results of the analysis are shown in table 5 below.

TABLE 5 post sequencing data analysis results

The same liquid phase hybridization capture probe is adopted in the experiment, and the effect of the self-made hybridization reagent and the washing reagent is evaluated under the same experiment condition. The result shows that all samples obtain good sequencing capture effect, the average sequencing depth is more than 15000X, the uniformity of each site is good, and the coverage rate reaches 100%. The result shows that the self-made reagent experimental group is obviously higher than the commercial reagent in the aspects of capture efficiency and data effectiveness, and the hybridization time is greatly shortened to 16h, so that the reagent and the method provided by the invention can achieve the effect that the detection result is better than the commercial kit.

in view of the present disclosure, it will be appreciated by those of ordinary skill in the art that the above-described embodiments are specific examples for implementing the invention, and that in practice, changes may be made in the details of operation without departing from the spirit and scope of the invention. The scope of the present invention is defined by the appended claims, but also includes equivalent variations of the content defined in the claims, for example, it is obvious to those skilled in the art that 3 ×, 4 ×, 5 ×, 6 ×, etc. stock solutions prepared according to the idea of the present invention and experimentally feasible are also unambiguously within the scope of the present invention as hybridization capture solutions.

Claims

1. A genome sequencing library hybridization kit comprises a hybridization capture kit and a washing solution kit,

The hybrid capture kit comprises 2 x concentration solution and 100% deionized formamide,

Wherein the 2 Xconcentration solution is 100mM sodium dihydrogen phosphate, 4 Xsodium citrate buffer solution, 2mM ethylene diamine tetraacetic acid, 2 XDenhardt solution, 2mg/ml Carrier RNA and 20 w/w% dextran sulfate,

Wherein the 4 Xsodium citrate solution is 0.6M sodium chloride, 0.06M sodium citrate, pH7.0;

And the 2 xdenhardt solution is 0.04% ficoll 400, 0.04% polyvinylpyrrolidone and 0.04% bovine serum albumin;

The wash solution kit comprises the following 5 wash buffer components:

3) washing solution II: 2 x sodium citrate solution;

4) washing solution III: 0.2 x sodium citrate solution; and

5) Streptavidin magnetic bead washing solution: 10mM Tris-HCl pH 7.5, 1mM EDTA and 2M NaCl,

Wherein the 2 x sodium citrate solution is 0.3M sodium chloride and 0.03M sodium citrate, ph7.0, and the 0.2 x sodium citrate solution is 0.03M sodium chloride and 0.003M sodium citrate, ph 7.0.

2. Use of the genomic sequencing library hybridization kit of claim 1 in probe hybridization capture enrichment of genomic DNA sequencing libraries.

3. A liquid phase hybridization capture method wherein the genomic sequencing library hybridization kit of claim 1 is used in the method.