True and false gene mutation analysis method based on high-throughput sequencing and application

Abstract

The invention relates to a true and false gene mutation analysis method based on high-throughput sequencing and application, and belongs to the technical field of bioinformatics. The true and false gene mutation analysis method comprises the following steps: acquiring difference sites in reference sequences of homologous true genes and false genes; comparing the NGS sequencing data with the difference sites to respectively obtain the true gene reads number and the false gene reads number corresponding to the same difference site, taking the ratio of the true gene reads number to the false genereads number of the same difference site as a judgment index, and carrying out mutation analysis and judgment on the true gene according to a predetermined strategy. According to the kit, mutation oftrue and false genes can be preliminarily screened, genes which possibly have problems are found out, and judgment is carried out in combination with clinical actual conditions. MLPA or sanger sequencing experiments carried out on genes one by one are avoided, and the experiment cost and time are greatly saved.

Description

technical field [0001] The invention relates to the technical field of bioinformatics, in particular to a high-throughput sequencing-based method for analyzing true and false gene mutations and its application. Background technique [0002] Pseudogenes, also called pseudogenes, are nonfunctional residues of gene families formed during evolution. It is similar to a normal gene, but the DNA sequence pseudogene that loses its normal function can be regarded as a non-functional genomic DNA copy that is very similar to the coding gene sequence in the genome. Generally, it is not transcribed and has no clear physiological meaning. [0003] However, there are some genes in the human genome that have highly homologous pseudogenes, such as homologous gene pairs such as SMN1 / SMN2 and CYP21A2 / CYP21A1P. When using NGS sequencing, since the alignment of reads is based on the principle of optimal matching, when the bases in the true gene are mutated to the bases of the pseudogene, these ...

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Problems solved by technology

If there is no pseudogene, the reads near this position will be compared to the real gene (although there is a mismatch, but it is still the best match), because C and T do not match, after comparison, you can find C at this position >T mutation; however, due to the existence of the pseudogene SMN2 on the genome, it is the best match for the reads near this place to be compared to the pseudogene, so the reads with this mutation actually cannot be compared to the real gene , it is impossible to find the mutation of C>T at this position on the true gene

[0005] At present, there is no good method for NGS sequencing to deal with pseudogenes. Generally, additional MLPA is required or the true gene is completely amplified and then analyzed by sanger sequencing. The cost is high and additional experimental time is required.

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