Optimized method for constructing RNA high-throughput sequencing library and application of optimized method

Abstract

The invention belongs to the field of molecular biological techniques and application, and particularly relates to an optimized method for constructing an RNA high-throughput sequencing library and application of the optimized method. The optimized method for constructing the RNA high-throughput sequencing library is characterized in that the method adopts RNase III to break to-be-sequenced RNA, adopts RNA ligase to connect linkers and adopts reverse transcriptase and a high-fidelity DNA polymerase system to conduct one-step reverse transcription and labeling PCR to construct the high-throughput sequencing library. The method can be applied to construction of the RNA high-throughput sequencing library. According to the optimized method for constructing the RNA high-throughput sequencing library, RNA is directly adopted to add the linkers onto single-strands, the step of random primer amplification is omitted, the influences of random primers can be eliminated, and more comprehensive RNA information is obtained; and an one-step method is adopted to conduct reverse transcription and labeling PCR, intermediate steps are reduced, and the preparation efficiency of the RNA library and the sequencing quality are enhanced effectively.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an optimized method for constructing an RNA high-throughput sequencing library and its application. Background technique [0002] Next-generation sequencing (NGS) represented by the Illumina sequencing platform has been widely used in the fields of DNA sequencing and RNA sequencing. RNA-seq can sequence the RNA sequences of mRNA, small RNA, non-coding RNA, RNA virus, etc. using high-throughput sequencing technology in one experiment. [0003] Usually, the construction of an RNA sequencing library generally includes the following steps: mechanically or enzymatically fragmenting the RNA fragments to a certain length, purifying the fragmented RNA, reverse transcription using random primers, synthesizing the second strand of cDNA, repairing the end and adding A Adapters, purification, index PCR amplification and index PCR product purification. Such a library construction ...

AI Technical Summary

Problems solved by technology

Such a library construction method includes three tedious purification steps, causing a great waste of experimental reagents, consumables, time and manpower

At the same time, random primers need to be used in the process of building a library, and random primers sometimes cause an imbalance in reverse transcription and bring certain deviations to the sequencing results

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