Method and kit for simultaneously constructing sequencing library by DNA and RNA

Abstract

The invention discloses a method and a kit for simultaneously constructing a sequencing library by DNA and RNA. The method comprises the following steps: (1) carrying out reverse transcription on RNAin a reaction substrate to form RNA / cDNA composite double strands; (2) directly adding Tn5 transposase and a fragmentation buffer solution into a mixture of a reverse transcription product cDNA / RNA composite double strand and a DNA double strand for fragmentation, and adding a part of linker sequence to the 5' end of the double strands during fragmentation; (3) strand displacement and library amplification: adding an amplification buffer solution and a strand displacement and amplification enzyme mixture into the obtained product, and carrying out PCR amplification; and (4) carrying out magnetic bead purification to obtain a sequencing library of the original DNA / RNA. According to the method, the library building steps are greatly reduced, the library building time is shortened, the library output and offline data quality is improved, and more real information is helped to be obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and a kit for simultaneously constructing a sequencing library from DNA and RNA. Background technique [0002] High-throughput sequencing technology, also known as second-generation sequencing technology, can be abbreviated as NGS, which refers to the technology of sequencing hundreds of thousands to several million DNA molecules in parallel at a time, and the length of the sequence is generally short. [0003] High-throughput sequencing mainly includes the following processes: sample collection and nucleic acid extraction, target sequence enrichment and sequencing library construction, sequencing and result analysis. The library construction includes DNA library construction and RNA library construction. [0004] DNA library construction generally involves randomly interrupting the DNA, adding an A linker after the end is repaired, and performing PCR amplification and qual...

AI Technical Summary

Problems solved by technology

[0006] At present, for DNA and RNA library construction at the same time, the RNA is generally mixed with DNA after reverse transcription and second-strand synthesis, and then the library is constructed through the normal DNA library construction process. The details are as follows: figure 1 As shown (taking 8 hours), that is, the traditional DNA library construction and RNA library construction are both for specific nucleic acid library construction, and it is impossible to build a real DNA and RNA library together. This method has a long operation process, cumbersome steps, and consumes very long

[0007] The key step for simultaneous construction of DNA and RNA libraries is the transposase breaking step. The efficiency of Tn5 transposase breaking cDNA hybrid strands is low, and the inactivation of the transposase is not complete during the termination reaction, and the transposase sticks to the DNA. On the other hand, affecting the subsequent amplification will have a greater impact on the quality and speed of library construction

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