Rapid single cell library building method

Abstract

The invention provides a rapid single cell library building method which comprises the following steps: 1) splitting a single cell, and releasing whole genome DNA in the single cell; 2) adding Tn5 transposase into the whole genome DNA obtained in the step 1), and cutting the DNA into DNA fragments; 3) performing PCR amplification on the obtained DNA fragments, introducing an index bar code in the amplification process, adding a cell index bar code for each single cell, and a PCR amplification reagent further comprising a fragmentation stop solution to stop fragmentation; and 4) purifying the amplified DNA fragment to obtain the single cell genome library. The invention also provides a rapid single-cell whole-genome library building method integrated on the digital micro-fluidic chip, and on the basis of the method, the method is integrated on the digital micro-fluidic chip to realize automatic library building. According to the method disclosed by the invention, a pre-amplification step is omitted, but amplification bias can still be reduced, a better sequencing coverage rate is obtained, favorable information is provided for CNV and SNV analysis, and the sequencing time, reagent cost and labor cost are greatly shortened.

Description

technical field [0001] This application relates to the field of single-cell sequencing, in particular to the field of single-cell whole-genome library construction. Background technique [0002] Single-cell sequencing technology refers to a new technology for high-throughput sequencing analysis of genome, transcriptome, and epigenome at the level of a single cell. It can reveal the gene structure and gene expression state of a single cell, reflect the heterogeneity among cells, and play an important role in the fields of tumors, developmental biology, microbiology, and neuroscience. In 2013, "Science" magazine ranked single-cell sequencing as the top six fields worth paying attention to in the year, and believed that single-cell sequencing technology will change many fields in the biological and medical fields. The clinical value of single-cell sequencing lies in the analysis of rare cancer cells in clinical samples, early diagnosis of tumors, discovery of new drug targets,...

AI Technical Summary

Problems solved by technology

[0006] Sequencing results with higher coverage depth and breadth can be obtained through pre-amplification, but it will also lead to amplification bias and loss of coverage, and amplification bias reduces coverage uniformity, thus covering up the detection of CNV (Copy numbervariant) , and the replication error of the polymerase will lead to wrong detection of SNV (Single-nucleotide variant)

Moreover, most WGA methods have disadvantages such as cumbersome procedures, high requirements on the amplification environment, easy pollution, time-consuming, expensive amplification reagents, etc., which greatly increase the time of sequencing sample preparation and the cost of reagents.

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