An Absolute Quantitative Transcriptome Library Construction Method Based on Unique Recognition Sequences

A technology for sequence recognition and library construction, which is applied in chemical libraries, biochemical equipment and methods, and microbial assay/inspection, etc. It can solve the problems of inability to accurately quantify transcripts, few steps for library construction, and low initial RNA quantity. Achieve the effects of reducing the initial amount of RNA library construction, increasing the speed of library construction, and improving the efficiency of linker ligation

Active Publication Date: 2021-10-22
武汉康测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method provided by the invention has the characteristics of high efficiency of library construction, few steps of library construction, and low initial amount of RNA, especially can completely solve the problem that the existing technology cannot accurately quantify transcripts

Method used

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  • An Absolute Quantitative Transcriptome Library Construction Method Based on Unique Recognition Sequences
  • An Absolute Quantitative Transcriptome Library Construction Method Based on Unique Recognition Sequences
  • An Absolute Quantitative Transcriptome Library Construction Method Based on Unique Recognition Sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] [Example 1] Construction of Absolute Quantitative Transcriptome Library Based on Unique Recognition Sequence

[0045] 1. mRNA capture

[0046] 1. Extract high-quality total RNA from control cells (NC) and GAS5 knockdown Hela cells (Si_GAS5) and capture mRNA from them. While adopting the technical scheme of the present invention to construct the transcriptome library, conventional transcription is performed to construct the library.

[0047] 2. In a Nuclease-free PCR tube, dissolve 0.1-4 μg of total RNA in Nuclease-free H 2 O, to a total volume of 50 μL, keep on ice for later use. Pipette 50 μL of washed magnetic beads (Roche, 11787896001) to mix with the RNA sample, pipette and mix well, then put it into a PCR machine and incubate at 65°C for 5min, then at 20°C for 5min. Place the sample on the magnetic stand for 5 minutes (until the solution is clarified), carefully remove the supernatant; take the sample out of the magnetic stand, add 200 μL Washing Buffer (Roche, ...

Embodiment 2

[0102] [Example 2] Sequencing data analysis process

[0103] S1: Perform quality control on raw data, remove low-quality bases and cut off corresponding adapters;

[0104] S2: Analyze the UID sequence on the reads, and regard the reads under the same UID sequence as a cluster (cluster);

[0105] S3: According to the above principles, since the reads under the same UID sequence come from the same molecule, the reads under each cluster are assembled consistently to form a consistent read. Such as Figure 4 As shown, in the process of assembly, the function of deduplication is actually realized, that is, molecules from the same source are finally merged into one sequence. At the same time, the purpose of error correction is also achieved, because the erroneous bases introduced by the reads under the same cluster during PCR amplification or sequencing on the machine will be corrected based on the consensus sequence of multiple reads. The resulting results are used as the final ...

Embodiment 3

[0121] [Example 3] Library construction and sequencing with different starting quantities for library construction

[0122] Extract the total RNA of Hela cells, use 100ng, 500ng, and 1ug respectively as the initial amount of library construction, build the library according to the steps of [Example 1], and use 1% agarose gel electrophoresis to detect the constructed library, as shown in Image 6 . Sequencing data analysis was performed according to the steps of [Example 2]. Correlation analysis was carried out on the sequencing results of different initial amounts of library construction, and the Pearson correlation coefficient R 2 The closer to 1, the higher the similarity of RNA expression patterns. The correlations of sequencing results with different starting amounts for library construction were all above 0.97. Such as Figure 7 shown.

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Abstract

The invention discloses a method for constructing an absolute quantitative transcriptome library based on a unique recognition sequence. Using the fragmented mRNA as a template, the first cDNA strand is synthesized under the action of reverse transcriptase using a primer pool with a universal linker sequence, and a unique recognition UID sequence is added to the 3' end of the synthesized cDNA by an enzymatic reaction. Build a library linker so that each cDNA has a unique sequence tag; finally, use a universal library linker to perform PCR amplification to obtain an RNA library. For the first time, the present invention uses the splint ligation method to construct an RNA library based on single-stranded cDNA. At the same time, the UID sequence is used to accurately restore the cDNA composition before PCR amplification, which can realize accurate quantification of transcripts; the present invention uses single-stranded cDNA as a raw material for library construction. The step of second-strand synthesis is omitted, the template loss rate is reduced, cost and time are saved, and the existing technology can only relatively quantify transcripts.

Description

technical field [0001] The invention belongs to the technical field of gene sequencing, and in particular relates to a method for constructing an absolute quantitative transcriptome library based on a unique recognition sequence UID. Background technique [0002] mRNA accounts for about 3% of the total RNA in cells, but because it is finally translated into protein and participates in the phenotypic composition of species, it has always been the focus of research. In the past ten years, the rapid development of next-generation sequencing has promoted the continuous progress of life sciences. With the large-scale application of next-generation sequencing technology, researchers have a deeper understanding of the field of life sciences. Compared with the genome, the transcriptome includes time and space limitations, and the transcriptome is much smaller than the genome. Under the same coverage multiple, the amount of sequencing data required is also much smaller than the amoun...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/6869
CPCC12N15/11C12Q1/6869C40B50/06C12Q2537/165
Inventor 吴启家王琳蒋菁菁
Owner 武汉康测科技有限公司
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