A method and kit for accurately quantifying next-generation sequencing libraries with different GC contents using qPCR

Abstract

The invention discloses a kit and a method for precise quantification of next-generation sequencing libraries different in GC content by qPCR (quantitative polymerase chain reaction). The kit for precise quantification of the next-generation sequencing libraries different in GC content by qPCR comprises a plurality of standard samples different in GC content, library dilution liquid, qPCR premixed liquid, primers and ddH2O, wherein nucleotide sequences of the primers are as shown in SEQ ID NO:1 and SEQ ID NO:2. The invention further provides the method of adopting the kit for precise quantification of the next-generation sequencing libraries different in GC content by qPCR. The kit and the method have advantages of precise quantification, convenience in use, wide application range, high selectivity, high sensitivity, high specificity and high stability and provide a guarantee for high-quality high-efficiency sequencing.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, that is, the category of next-generation sequencing (NGS). More specifically, it relates to a method and a kit for accurately quantifying next-generation sequencing libraries with different GC contents by using qPCR. Background technique [0002] With the continuous advancement of science and technology, traditional Sanger sequencing can no longer fully meet the needs of research and applications. For genome sequencing, a sequencing technology with lower cost, faster speed, and higher throughput is required—the second-generation sequencing technology (NGS) ) came into being. Quantitative analysis and quality evaluation of the sequencing library are very important for obtaining high-quality nucleic acid sequencing data. To control the loading of the sequencing library on the NGS sequencer and ensure the quality of the sequencing data, it is necessary to accurately quantify the DNA sequen...

AI Technical Summary

Problems solved by technology

For the quantification of libraries with different GC content, especially the high GC / AT library, in order to ensure the accurate quantification of qPCR method, at present, the preference of DNA polymerase to avoid PCR amplification, optimization of reaction system (adding GC buffer) and optimization of reaction are mainly used. conditions (increase the pre-denaturation temperature), etc., for example, the KAPA SYBR FAST DNA polymerase included in the mainstream product KAPA LibraryQuantification Kits on the market claims that it can obtain similar amplification efficiency for DNA fragments with high GC / AT, but our Multiple comparison experiments were performed to detect the same concentration of standard substances with different GC contents, and it was found that the Ct values ​​were significantly different ( figure 1 ), which shows that it still cannot guarantee the same amplification efficiency, avoids the amplification bias of the template, and has a lower amplification efficiency for GC / AT-rich standards

Therefore, current methods cannot accurately quantify libraries with different GC contents if only one GC content standard is used

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