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RNA and DNA co-construction library sequencing method and library construction instrument

A sequencing and instrument technology, applied in the field of gene sequencing library, to improve the efficiency of sample use and library construction, save time, and avoid waste of consumables

Inactive Publication Date: 2021-03-02
深圳易倍科华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to propose a RNA and DNA co-construction library sequencing method to solve the problem of simultaneous library construction of RNA and DNA mixtures

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  • RNA and DNA co-construction library sequencing method and library construction instrument
  • RNA and DNA co-construction library sequencing method and library construction instrument
  • RNA and DNA co-construction library sequencing method and library construction instrument

Examples

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Embodiment 1

[0045] see figure 1 , Embodiment 1 of the present invention provides a RNA and DNA co-construction library sequencing method, the method comprising:

[0046] Step S10: fragment the RNA in the DNA / RNA total nucleic acid sample to be tested to obtain RNA fragments; specifically, the random fragmentation includes any one of enzymatic fragmentation, mechanical fragmentation and chemical ion fragmentation one or more species. Specifically in this embodiment one, using Mg 2+ Interrupt method, using Mg 2+ Reagents can use commercial products. The buffer solution final concentration used among the present invention is as follows: 50-100mM Tris-HCl (PH 7.5), 2.5 μ M-10 μ M MgCl 2 , 25 μM-100 μM KCl, 1%-3% DMSO, 0.5-2% PEG, 1-10 μg / ml BSA, 1-500 ng RNA. After preparing the above system, according to the fragment size required by the experiment, react at 85-94°C for 5-10min. Among them, BSA is bovine serum albumin, Tris-HCl is tromethamine hydrochloride, MgCl 2 is magnesium chlori...

Embodiment 2

[0070] Example 2 of the present invention detects genes related to lung cancer, including RNA fusion and mutation detection for ALK, NTRK, RET, ROS1 and other genes, and mutation detection for BRAF, EGFR, ERBB2, KRAS, MET, NRAS and other genes. Sequenced using the illumina Novaseq platform. The probe pool (Panel) design is aimed at the whole exon design of the above genes, and the probes synthesized by Integrated DNA Technologies in the United States (other equivalent probes are also available). The specific IDT probes used are shown in Table 6 below:

[0071] Table 6:

[0072] Gene Detect mutation type exon coverage ALK Single point mutation / indel / fusion full coding sequence BRAF Single point mutation / indel / full coding sequence EGFR Single point mutation / indel / copy number variation full coding sequence ERBB2 Single point mutation / indel / copy number variation full coding sequence KRAS Single point mutation / indel full c...

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Abstract

The invention provides an RNA and DNA co-construction library sequencing method, which comprises the following steps: breaking RNA in a to-be-detected DNA / RNA total nucleic acid sample to obtain an RNA fragment; carrying out reverse transcription on the RNA fragment by using a random primer to obtain a cDNA fragment, and preferably using a random primer sequence containing a molecular tag UMI R todistinguish between the cDNA fragment and the first DNA fragment present in the total nucleic acid; adding magnetic beads for primary purification, removing the magnetic beads to obtain supernatant DNA, and transferring the supernatant DNA to a new test tube; breaking the supernatant DNA subjected to the first purification by a mechanical breaking method or an enzyme breaking method to obtain a second DNA fragment required by sequencing; carrying out first joint connection and second joint connection, and carrying out purification treatment to obtain a first product; and carrying out PCR amplification reaction on the first product, and carrying out fourth purification treatment to complete library building. According to the technical scheme provided by the invention, DNA and RNA are subjected to library building at the same time, and the sample use efficiency and the library building efficiency are improved.

Description

【Technical field】 [0001] The invention relates to the technical field of gene sequencing libraries, in particular to an RNA and DNA co-construction and sequencing method and a library construction instrument. 【Background technique】 [0002] Next-generation sequencing (NGS), third-generation sequencing, and even fourth-generation sequencing technologies have been applied in the fields of detection, diagnosis, and scientific and technological research, but how to build and sequence libraries for total nucleic acids (DNA and RNA) is currently a major challenge. Due to the differences in the physical and chemical properties of DNA and RNA, conventional gene sequencing technology can only target single nucleic acid DNA or RNA. Relatively speaking, the development of a single DNA sequencing technology is relatively mature, while the RNA sequencing technology is relatively complicated. The common method of DNA library construction is double-strand library construction. After the D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 徐飞岳缑灵山管少华黎亿爱
Owner 深圳易倍科华生物科技有限公司