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A method and kit for constructing a library for whole-genome high-throughput sequencing

A technology for sequencing libraries and whole genomes, applied in chemical libraries, combinatorial chemistry, recombinant DNA technology, etc., can solve the problems of high sequencing cost and unusable redundant sequencing data, and achieve the effect of simple operation and short time-consuming

Active Publication Date: 2021-04-06
BERRYGENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The higher the redundancy, the more sequencing data cannot be used, and the higher the sequencing cost

Method used

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  • A method and kit for constructing a library for whole-genome high-throughput sequencing
  • A method and kit for constructing a library for whole-genome high-throughput sequencing
  • A method and kit for constructing a library for whole-genome high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Using the standard cell line NA12878 genomic DNA, using a common Y-type connector (TruSeq connector), a Y-type reverse connector according to the present invention, and a novel connector combination according to the present invention (Y-type reverse connector + high GC splint) respectively linker) to construct a PCR-free library, and use the PCR library as a control. Sequencing on the machine, data analysis, and comparing the sequencing data of the PCR-free library and the PCR library constructed by three different adapters.

[0093] According to the structure of the Y-type reverse joint of the present invention, such as image 3 Shown; According to the structure of the novel joint combination of the present invention (Y type reverse joint + high GC splint joint) such as Figure 4 shown.

[0094] In this example, NA12878 gDNA was used as a sample to construct PCR-free whole-genome high-throughput sequencing using common Y-type connectors, Y-type reverse con...

Embodiment 2

[0110] Example 2: Human genomic DNA was used to construct a PCR-free library using common Y-joints (TrueSeq joints), Y-type reverse joints, combinations of common Y-joints and high GC splint joints, and novel joints of the present invention, respectively. Sequence together with the phix library, analyze the number of phix sequences detected in the library, calculate the crosstalk ratio of the index, and compare the redundancy under the same amount of data at the same time.

[0111] This example uses phix's principle of testing crosstalk ratios: phix library inserts are derived from viral genomic DNA. Its gene sequence has been precisely known, and its GC ratio is about 40, which is close to the GC ratio of the human genome. Its gene sequence is far from that of human beings and does not contain an index. Therefore, the library to be tested and phix were sequenced together on the computer, the number of phix sequences in the library was analyzed, and the ratio of the sequence ...

Embodiment 3

[0117] Example 3: Using different input amounts of templates to construct a library, perform sequencing on a computer, analyze data, and compare the sequencing data quality and performance analysis results of different input amounts.

[0118] In this example, using the novel linker combination according to the present invention, NA12878 genomic DNA was used as a sample to construct genome-wide high-throughput sequencing libraries with inputs of 200 ng and 300 ng respectively, and the library was subjected to 150PE paired-end sequencing on NovaSeq. Bioinformatics analyzes the sequencing results and analyzes the library quality of libraries constructed with different adapters. The following is the specific scheme.

[0119] Step 1: Prepare the reaction mixture shown in Table 8, prepare four tubes, two tubes with a DNA input volume of 200ng, and the other two tubes with a DNA input volume of 300ng, and then run the interruption, end filling and addition shown in Table 9 together A...

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Abstract

The invention discloses a method for constructing a whole-genome high-throughput sequencing library, comprising the following steps: 1) extracting sample gDNA; 2) enzymatically cleaving the sample gDNA into fragments, filling in the ends, and adding A to obtain the added A 3) Ligate the A-added gDNA with an adapter combination to obtain a ligation product, the adapter combination includes two parts: a Y-shaped reverse adapter and a high GC splint adapter; 4) Purify the ligation product, obtaining a purified product; and 5) performing fragment screening on the purified product to obtain a sequencing library. The invention also discloses a kit for constructing a whole genome high-throughput sequencing library.

Description

technical field [0001] The invention relates to a method and a kit for constructing a library of whole genome high-throughput sequencing. More specifically, the present invention relates to a method and kit for constructing a genome-wide high-throughput sequencing library that can reduce redundancy and sequence tag (index) crosstalk. Background technique [0002] Whole Genome Sequencing (WGS) is the use of high-throughput sequencing platforms to sequence the whole genome of different human individuals or populations, and perform bioinformatics analysis at the individual or population level. It can comprehensively mine genetic variation at the DNA level, providing important information for screening disease-causing and susceptibility genes and studying pathogenesis and genetic mechanisms. Compared with whole-exome sequencing, whole-genome sequencing has its unique advantages because the results contain complete and rich information and can obtain more information that cannot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N15/11
CPCC40B50/06C12N15/11C12N15/1093C12Q1/6806C12Q2521/301C12Q2525/191C12Q1/6869
Inventor 张建光陈迪商玲
Owner BERRYGENOMICS CO LTD
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