Library building method based on high-throughput sequencing

A high-throughput, sequencing technology, applied in the field of library construction based on high-throughput sequencing, can solve problems such as complex processes, complex operations, and greater impact on the capture efficiency of target products, achieving good connection effects and ensuring specificity

Active Publication Date: 2020-06-23
上海厦维医学检验实验室有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] This library construction method has the following disadvantages: (1) The library is dried and dehydrated with a vacuum pump of a special instrument, and then dissolved in a hybridization buffer, which easily leads to cross-contamination during vacuum treatment; (2) It is necessary to use a variety of elution buffers to capture the product Perform cleaning to remove non-specific binding. The operation of this step is relatively complicated, and the reaction system is sensitive to temperature. The proficiency of the experimenter has a great influence on the capture efficiency of the target product; (3) if there are damaged double-stranded DNA molecules ( figure 1 B), it cannot be successfully amplified, resulting in the loss of the original DNA information; (4) If the nucleotide sample is RNA, it is necessary to reverse transcribe the RNA sample into cDNA first, and then obtain double-stranded DNA molecules through further PCR amplification. For subsequent connection and database building operations, the process is complicated and the cost is high
[0008] This library construction method has the following disadvantages: (1) It relies on the PCR amplification of the target region with clear forward and reverse primers, so it cannot be used for the detection of unknown types of fusion mutations; (2) if there is a damaged double-strand DNA molecules ( figure 2 B), it cannot be amplified smoothly, resulting in the loss of original DNA information
[0011] The above-mentioned method of building a library has the following disadvantages: (1) The use of biotin-labeled magnetic beads to enrich the ligation product leads to complex procedures, high cost, and the reaction system is sensitive to temperature. The operator's proficiency directly affects the concentration of the target product Capture efficiency; (2) If there are damaged double-stranded DNA molecules ( image 3 B), it cannot be successfully amplified, resulting in the loss of the original DNA information; (3) If the nucleotide sample is RNA, it is necessary to reverse transcribe the RNA sample into cDNA first, and then obtain double-stranded DNA molecules through further PCR amplification. For subsequent connection and database building operations, the process is complicated and the cost is high

Method used

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  • Library building method based on high-throughput sequencing
  • Library building method based on high-throughput sequencing
  • Library building method based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Prepare fragmented nucleotide samples.

[0053] 1. Genomic DNA sample:

[0054] (1) Open the M220 Focused-ultrasonicator (Covaris) and supporting software, put the Tube holderInsert into the water bath, and put the matching M220 holder XTU Insert microTUBE130μL ultrasonic tube loading sheet on it. Add about 15mL of ultrapure water until the surface of the Tube holder is submerged, and cover the safety cap until the water level indicator turns green, ready for use.

[0055] (2) Run the ultrasonic interruption program according to the following conditions.

[0056] Table 1 Instrument condition settings for ultrasonic crushing of genomic DNA samples

[0057] name condition Duty Factor 20% Peak 50 Cycle Burst 200 Volume 130μL Time 180s

[0058] (3) Transfer 125 μL of the interrupted product in step (1) to a 1.5 mL centrifuge tube, and add 250 μL AMPure XP Beads to the centrifuge tube, vortex and mix well, and incubate at ...

Embodiment 2

[0143] In order to illustrate that this library construction method can effectively carry out library construction and obtain high-throughput sequencing data enriched in target regions, the target regions (5860 bp in total) as shown in Table 4 below were selected for single-end nested multiplex PCR primer design and Synthesized to obtain the first gene-specific primers and the second gene-specific primers, a total of 77 pairs.

[0144] Table 4 Target region data of high-throughput sequencing

[0145]

[0146]

[0147] According to the specific operation steps in the above-mentioned Example 1, the nucleotide samples in the target region in Table 2 were constructed, and the results are as follows:

[0148] (1) The Agilent 2100 bioanalyzer system detects the size and distribution characteristics of nucleotide sample fragments for details. Image 6 As shown, the main peak size of the nucleotide sample fragment in this figure is 180bp.

[0149] (2) The size and distribution...

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Abstract

The invention discloses a library building method based on high-throughput sequencing. The library building method based on the high-throughput sequencing is applicable to fragmented nucleotide samples, such as fragmented genomic DNA / FFPE RNA / cfDNActDNA and the like. Furthermore, the fragmented nucleotide samples are subjected to modification, denaturation and joint connection, followed by enrichment of target regions by two specific PCR reactions, so as to obtain a sequencing library; and the sequencing library is subjected to quality control and quantitative analysis so as to carry out high-throughput sequencing. The invention further provides a simulated double-chain connection joint. The library building method based on the high-throughput sequencing is simple and convenient, and retains advantages of multiplex PCR library building methods based on amplicons; and moreover, the library building method based on the high-throughput sequencing can also be used for detecting unknownfusion, and is especially applicable for detecting nucleotide samples (e.g. cfDNA / ctDNA sample) with relatively high degree of fragmentation. The library building method based on the high-throughput sequencing is also feasible for building libraries of DNA molecules with damages. The library building method based on the high-throughput sequencing disclosed by the invention is capable of retaining all original nucleotide information to the maximum extent; and moreover, no two-chain synthesis is required when the method is adopted for carrying out RNA library building.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for building a library based on high-throughput sequencing. Background technique [0002] Today's human whole genome sequencing has made great progress, but there are still problems such as incomplete and accurate interpretation databases related to this technology, and high sequencing costs, so it is difficult for this technology to analyze some challenging genomic regions. At the same time, the high cost and complexity of this technology limit its application in specific genomic regions such as diseases and transformations. For this reason, a variety of methods for high-throughput sequencing library construction for enrichment of target regions for specific genomic regions have been widely developed and applied, thereby improving coverage, and achieving the purpose of simplifying the process and reducing costs. The library construction method of the present in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C40B50/06C12Q1/6876
CPCC12Q1/6869C40B50/06C12Q1/6876C12Q2535/122C12Q2525/191
Inventor 吴渊毕书琳李旭超李健鹏周文刚郑方克郑立谋
Owner 上海厦维医学检验实验室有限公司
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