High-abundance-RNA-removed sequencing library and construction method of sequencing library

Abstract

The invention belongs to the technical field of high-throughput sequencing, and discloses a high-abundance-RNA-removed sequencing library and a construction method of the sequencing library. The construction method comprises the following steps of: (1) extracting total RNA in a sample, and connecting all RNA with a 3'joint; (2) carrying out RNA fragmentation treatment; (3) connecting RNA with a 5'joint; (4) designing a DNA probe, and hybridizing the DNA probe with high-abundance RNA to form a DNA:RNA hybrid chain; (5) cutting the DNA:RNA hybrid chain to disconnect the high-abundance RNA from the 3'joint; and (6) carrying out reverse transcription and PCR amplification to obtain the high-abundance-RNA-removed sequencing library. The construction method can remove the high-abundance RNA in the high-throughput sequencing library, greatly reduces the proportion of the high-abundance RNA, has the advantages of simple experimental steps, low cost and good effect, can improve the quality of the large-scale RNA parallel sequencing library, and saves the cost.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a sequencing library for removing high-abundance RNA and a construction method thereof. Background technique [0002] Compared with the Sanger sequencing method (dideoxy termination method), which can only determine the sequence of several to hundreds of deoxyribonucleic acid (DNA) molecules each time, the new generation of high-throughput sequencing technology can detect millions of DNA each time Sequencing, this method uses a high-throughput method to connect sequencing adapters on heterogeneous DNA fragments (high-throughput DNA library construction), and uses biochemical reactions combined with imaging technology to sequence DNA tags. DNA libraries are mainly derived from two sources: 1) Sequencing adapters are directly added to both sides of short genomic DNA fragments; 2) After RNA fragmentation, it needs to be reverse-transcribed into cDNA, a...

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Problems solved by technology

[0008] Among the above four methods, the first method requires the synthesis of biotin-labeled nucleic acid probes. When there are many probes to be synthesized, the cost is relatively high. For example, it costs about 500 yuan to synthesize a primer containing biotin modification. If There are 20 high-abundance RNAs, and it takes nearly 10,000 yuan to synthesize 20 primers, and plant cells contain hundreds of high-abundance non-coding RNAs, so the cost is huge

The second method needs to design a specific single-stranded DNA probe and S9.6 antibody for immunoprecipitation, which is relatively expensive and the experiment is more complicated

The third method uses RNase H to cleave the RNA in the RNA:DNA hybrid strand, which requires the synthesis of longer single-stranded DNA probes, because if the entire RNA is not removed, there will be residual RNA cloned into the library, Affects quality, therefore uses a large number of DNA probes and RNase H, at higher cost

The fourth method uses DSN enzyme to cut the DNA strand in the RNA:DNA hybrid strand under specific conditions. Commercial DSN enzyme has a single source of supply and a high price

At the same time, for one treatment, specific enzyme cutting conditions need to be optimized, the operation is complicated and the specificity is difficult to control

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