Medium-small-length RNA high-throughput sequencing library building method and application thereof

Abstract

The invention provides a medium-small-length RNA high-throughput sequencing library building method and application thereof. Compared with an existing report, the technical scheme provided by the invention has the advantages that mRNA, lncRNA and snoRNA can be analyzed at the same time, and a repeatedly amplified RNA template rich in GC can also be analyzed; furthermore, the quantity of initial samples for library building is small; required time is short, deviation reduction is realized, the medium-small-length RNA high-throughput sequencing library building method is suitable for detecting whole cells, exosomes and even the latest technologies including HITS-CLIP / CLIP-seq, RIP-seq, ribosome profiling ribosome atlas analysis and the like; sequencing deviation is reduced, and full-length sequences of snoRNA and non-coding RNA of other structures can be obtained.

Description

[0001] This application claims the priority of the Chinese patent application submitted to the China Patent Office on December 28, 2018 with the application number 201811620297.6. The entire contents are incorporated by reference in this application. technical field [0002] The present invention relates to the field of high-throughput sequencing and library construction, and more specifically, to a high-sensitivity exosome fusion gene detection method and its application. Background technique [0003] All RNA-seq techniques require a cDNA synthesis step first: using reverse transcriptase (RT) to transcribe RNA into DNA, followed by high-throughput DNA sequencing. Existing RNA-seq techniques can be roughly divided into two types: [0004] The first one is suitable for the detection of mRNA and lncRNA, using oligo(dT) primers for reverse transcription to enrich poly(A)+RNA; the second is to use random primers for reverse transcription after removing highly abundant rRNA. Th...

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Problems solved by technology

[0008] However, the existing RNA-seq technology has high repeatability, but there will still be certain deviations due to different methods of RNA sample preparation, reverse transcription, and addition of adapters; in addition, because there are many structural proteins, such as tRNA and snoRNA, Heat resistant to traditional RNA-seq

[0009] Specifically, the existing technology has the following limitations: 1) mRNA, lncRNA, and small ncRNA sequencing cannot be performed simultaneously in the same RNA-seq reaction; 2) The relative fidelity of retroviral RT enzymes suitable for cDNA synthesis It is difficult to analyze RNA sequence polymorphisms, advanced structures, or GC-rich RNA; 3) Using RNA ligase or random hexamer primers to add adapters will produce bias

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