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Method for establishing sequencing library during detection of multiple samples and application of method

A method for constructing a sequencing library, which is applied in the field of constructing a sequencing library when multiple samples are detected, can solve the problems of unbalanced amplification among primers, large difference in data volume, high cost of primer synthesis, etc., to reduce the number of primers synthesized, reduce Effort, easy to promote the effect

Pending Publication Date: 2019-05-24
IPE BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For this reason, the present invention provides a method for constructing a sequencing library when multiple samples are detected, which is used to solve the large difference in the amount of data between samples in the prior art due to the classification of sequencing data and the identification of a single tag. The cost is high and the primers are cumbersome and error-prone, and it is not suitable to adjust the unbalanced amplification between primers

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  • Method for establishing sequencing library during detection of multiple samples and application of method
  • Method for establishing sequencing library during detection of multiple samples and application of method
  • Method for establishing sequencing library during detection of multiple samples and application of method

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Embodiment 1

[0034] This embodiment is a high-throughput sequencing library construction method for detecting 10 single nucleotide polymorphism sites in 64 copies of human genomic DNA. The construction method includes the following steps:

[0035] Step 1. Design upstream primer F1 and downstream primer R1 respectively according to the sequences of 10 specific single nucleotide polymorphism sites in genomic DNA, wherein the polymorphic sites, upstream primers and downstream primers are shown in Table 1;

[0036] Step 2, select 16 groups of sample labels (see Table 2), wherein 8 groups (001-008) are added as the first label in the middle of each upstream primer F1 and A adapter (such as figure 1 shown) to obtain upstream fusion primers; the other 8 groups (101-108) are added as second tags between each downstream primer R1 and P adapter to obtain downstream fusion primers; thus, 10 fusion primers of each group of sample tags can be obtained Primers, a total of 160; mix 10 fusion primers in e...

experiment example 1

[0045] A high-throughput sequencing method for detecting 10 single nucleotide polymorphism sites in 64 human genomic DNAs;

[0046] (1) Sample selection:

[0047] Select two human genomic DNA samples with unknown typing results, 64 each;

[0048] (2) Library construction

[0049] Using Example 1 and according to the existing ligation library construction method provided by Thermo Fisher, the two samples selected were respectively constructed for sequencing libraries;

[0050] (3) High-throughput DNA sequencing

[0051] High-throughput sequencing was performed using Ion Torrent PGM (purchased from Life Technologies) and supporting sequencing kits;

[0052] (4) Data processing

[0053] The analysis software SeqVision is used to complete, including three steps of data quality control, sequencing data classification and typing result conversion;

[0054] Quality control parameter setting: fragment length ≥ 60 bases;

[0055] Data classification parameter setting: Barcode Sor...

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Abstract

The invention discloses a method for establishing a sequencing library during detection of multiple samples and application of the method. The method includes the steps of firstly, designing upstreamprimers and downstream primers according to a genome DNA sequence; secondly, adding a connector A and a first label to each upstream primer to obtain an upstream fused primer, adding a connector P anda second label to each downstream primer to obtain a downstream fused primer, and mixing the upstream fused primer and the downstream fused primer to obtain a mixed fused primer; thirdly, preparing amultiplex PCR reaction system from the mixed fused primer, the genome DNA, a Tris-HCl buffer solution containing Mg2+, dNTPs and Taq enzymes and nuclease-free water; fourthly, conducting PCR reactionand purification on the obtained multiplex PCR reaction system to obtain the multi-sample sequencing library. In the establishing method, the samples are recognized through the combinations of the different labels on the upstream primers and downstream primers, and the synthesis number of the primers can be greatly reduced; in addition, the amplification frequency can be reduced, and the data imbalance between the samples is effectively reduced.

Description

technical field [0001] The invention relates to the technical fields of biomedicine, forensic science, criminal investigation and physical evidence identification, in particular to a method for constructing a sequencing library when detecting multiple samples and its application. Background technique [0002] High-throughput sequencing technology has developed rapidly since 2010. Due to the large amount of data that can be obtained by this technology, according to different project needs, it can meet the locus typing requirements of dozens or even hundreds of samples. In order to distinguish samples, when constructing the library, a sample tag (barcode) was added before the sequence of each sample, and the sequence data was divided into each sample by using the recognition sequence during data analysis. [0003] Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) refers to the polymorphism of nucleic acid sequence caused by the change of a single nucleotide ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6806
Inventor 周骋
Owner IPE BIOTECHNOLOGY CO LTD