Full exon sequencing (WES 1000 g) was performed by Agilent's liquid chip capture system. Genomic DNA extracted from peripheral blood for each sample was fragmented to an average size of 180–280 bp and subjected to DNA library creation using established Illumina paired-end protocols. The Agilent SureSelect Human All ExonV6 Kit (Agilent Technologies, Santa Clara, CA, USA) was used for exome capture according to the manufacturer’s instructions. The Illumina Novaseq 6000 platform (Illumina Inc., San Diego, CA, USA) was utilized for genomic DNA sequencing in Genechem Bioinformatics Technology Co., Ltd (Beijing, China) to generate 150-bp paired-end reads with a minimum coverage of 10 × for ~ 99% of the genome (mean coverage of 100 ×). After sequencing, base call files conversion and demultiplexing were performed with bcl2fastq software (Illumina). The resulting fastq data were submitted to in-house quality control software for removing low quality reads, and then were aligned to the reference human genome (hs37d5) using the Burrows-Wheeler Aligner (bwa), and duplicate reads were marked using Sambamba tools. ANNOVAR software was used to annotate the variants.
Filtering of rare variants was performed as follows: (1) variants with a MAF less than 0.01 in 1000 genomic data (1000g_all), esp6500siv2_all, gnomAD data (gnomAD_ALL and gnomAD_EAS) and in house Genechem-Zhonghua exome database from Genechem; (2) Only SNVs occurring in exons or splice sites (splicing junction 10 bp) are further analyzed since we are interested in amino acid changes. (3) Then synonymous SNVs which are not relevant to the amino acid alternation predicted by dbscSNV are discarded; The small fragment non-frameshift (< 10 bp) indel in the repeat region defined by RepeatMasker are discarded. (4) Variations are screened according to scores of SIFT, Polyphen, MutationTaster and CADD software. The potentially deleterious variations are reserved if the score of more than half of these four software support harmfulness of variations. Sites (> 2 bp) did not affect alternative splicing were removed.
Filtering of rare variants was performed as follows: (1) variants with a MAF less than 0.01 in 1000 genomic data (1000g_all), esp6500siv2_all, gnomAD data (gnomAD_ALL and gnomAD_EAS) and in house Genechem-Zhonghua exome database from Genechem; (2) Only SNVs occurring in exons or splice sites (splicing junction 10 bp) are further analyzed since we are interested in amino acid changes. (3) Then synonymous SNVs which are not relevant to the amino acid alternation predicted by dbscSNV are discarded; The small fragment non-frameshift (< 10 bp) indel in the repeat region defined by RepeatMasker are discarded. (4) Variations are screened according to scores of SIFT, Polyphen, MutationTaster and CADD software. The potentially deleterious variations are reserved if the score of more than half of these four software support harmfulness of variations. Sites (> 2 bp) did not affect alternative splicing were removed.
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