Method for establishing single cell transcriptome sequencing library and application of method

Abstract

The invention discloses a method for establishing a single cell transcriptome sequencing library and application of the method. The method comprises the following steps: performing reverse transcription on RNA in a single cell, thus obtaining cDNA; performing pre-amplification on cDNA by using an amplification primer, thus obtaining amplified cDNA; and performing fragmentation library construction on the amplified cDNA, thus obtaining a transcriptome sequencing library of the single cell, wherein dTTP in the amplification primer is substituted by dUTP. According to the method disclosed by the invention, as the dTTP in the pre-amplification primer is substituted by dUTP, fragments containing the pre-amplification primer in a jointed fragment can be interrupted in an enzymic digestion step after the step of joint connection, and are removed in later high-temperature pre-denaturation and denaturation steps; furthermore, the amplification of fragments with the pre-amplification primer can be reduced, the ratio that the proportion of data with pre-amplification primer pollution in the obtained sequencing data is greatly reduced, and the effective data amount of the obtained data is greatly increased.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, in particular to a method for constructing a single-cell transcriptome sequencing library and its application. Background technique [0002] For single-cell transcriptome sequencing, the construction of transcriptome sequencing library is the main difficulty, because the mRNA content in a single cell is as low as 10pg, which cannot be constructed by conventional RNA transcriptome library construction methods. Therefore, the products currently on the market that can use such a small amount of mRNA for single-cell transcriptome sequencing library construction generally use Illumina's DNA library construction kit (illumina Nextera XT DNA sample preparation kit) after obtaining pre-amplified cDNA. [0003] The kit of Illumina uses a chemical method to fragment the sample, that is, uses a transposase to fragment the cDNA to build a library. After the single cell is lysed, the mRNA is reverse...

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Problems solved by technology

[0005] However, a pre-amplification step is required before fragmentation, so that the amount of cDNA can meet the requirements for library construction. However, this physical fragmentation method fragments the cDNA, which will make the constructed library contain pre-amplification. Primer pollution, accounting for about 20% of the total data, resulting in waste of data

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