Preparation method of capture probe for targeted sequencing of novel coronavirus virus SARS-CoV-2 genome

Abstract

The invention relates to genomic targeted sequencing. The preparation method of the capture probe for targeted sequencing of the novel coronavirus virus SARS-CoV-2 genome comprises the following steps: downloading an SARS-CoV-2 virus reference genome sequence NC _ 045512.2 from an NCBI database, adopting an imbricated coverage design strategy, and screening probes with the length of 75-120nt according to parameters such as GC content, a Tm value and homology, carrying out quantitative evaluation on the probe library, filtering out unqualified probe sequences, and leaving 249 probes to cover the whole genome of the novel coronavirus virus; preparing a DNA templates of 249 probe libraries, performing PCR amplification, and purifying PCR products; carrying out enzyme digestion on the purifiedPCR product and smoothly supplementing the tail end of T4 DNA polymerase, and digesting a single strand with 5'end phosphorylation in the double-stranded DNA by using Lambda exonuclease to obtain a single-stranded DNA with a biotin label; and purifying the single-stranded DNA with the biotin label by a magnetic bead enrichment method to obtain the modified probe library. According to the method,the sequencing data volume is reduced, the data analysis process is simplified, and the sensitivity and accuracy of the NGS method for detecting the novel coronavirus virus are improved.

Description

Technical field [0001] The present invention relates to targeted sequencing of the genome, in particular to targeted NGS sequencing for the genome of the new coronavirus SARS-CoV-2. Background technique [0002] In the field of nucleic acid detection methods, after PCR and first-generation sequencing, next-generation sequencing (NGS) is widely used in various application fields of nucleic acid molecular diagnosis. With the advancement of technology, the data throughput of NGS is getting higher and higher, and the cost of sequencing is getting lower and lower. However, whole-genome sequencing with a large sample size of different species, even a single-sample whole-genome sequencing, is relatively difficult in practice, regardless of cost or data analysis. [0003] Targeting the target area and then NGS sequencing (such as figure 1 ), the targeted enrichment of the target gene or DNA fragments of the genomic region from the whole genome before NGS sequencing greatly reduces the cos...

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Problems solved by technology

[0006] 2) Usually, RT-PCR primers are designed to amplify the target regions of the SARS-CoV-2 genome are conservative sequences, but the new coronavirus is an RNA virus, once mutation and recombination occur, RT-PCR primers are difficult to effectively amplify;

[0007] 3) The virus load of the new crown patients with typical symptoms or asymptomatic carriers is unstable. Even if the test result obtained by RT-PCR method is positive, bacterial infection or viral co-infection cannot be ruled out;

[0010] For biological samples from a large number of sources, if the pathogen load is low, after effectively removing the data of human hosts, it is still difficult for NGS to guarantee the sensitivity of clinical detection

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