title: Whole exon sequencinghuman peripheral blood cells
design: DNA Library PreparationFor exome sequencing, we fragmented 13 g ofgenomic DNA, extracted from eachsample, to an average size of 180 bp with a Bioruptorsonicator (Diagenode). Paired-end sequencinglibraries then were prepared using a DNA sampleprep reagent set 1 (NEBNext). Library preparationincluded end repair, adapter ligation and PCRenrichment, and was carried out as recommendedby Illumina protocols.Targeted genes enrichment and sequencingThe amplified DNA was captured use GenCap Deafness capture kit. The DNA probes were designed to tile along the exon regions and the known non-exon pathogenic region of human genes. The capture experiment was conducted according to manufacturers protocol. In brief, 1g DNA library was mixed with Buffer BL and GenCap gene panel probe , heated at 95C for 7 min and 65C for 2 min on a PCR machine; 23l of the 65C prewarmed Buffer HY was then added to the mix, and the mixture was held at 65C with PCR lid heat on for 22 hours for hybridization. 50 l MyOne beads was washed in 500L 1X binding buffer for 3 times and resuspended in 80l 1X binding buffer. 64 l 2X binding buffer was added to the hybrid mix, and transferred to the tube with 80l MyOne beads. The mix was rotated for 1 hour on a rotator. The beads were then washed with WB1 buffer at room temperature for 15 minutes once and WB3 buffer at 65C for 15 minutes three times. The bound DNA was then eluted with Buffer Elute. The eluted DNA was finally amplified for 15 cycles using the following program: 98C for 30 s (1 cycle); 98C for 25 s, 65C for 30 s, 72C for 30 s (15 cycles); 72C for 5 min (1 cycle). The PCR product was purified using SPRI beads according to manufacturers protocol. The enrichment libraries were sequenced on Illumina HiSeq X ten sequencer for paired read 150bp.Bioinformatics analysisAfter sequencing, the rawdata were saved as a FASTQ format, then followed the bioinformatics analysis: First, Illumina sequencing adapters and low quality reads (<80bp) were filtered by cutadapt. After quality control, the clean reads were mapped to the UCSC hg19 human reference genome using BWA. Duplicated reads were removed using picard tools and mapping reads were used for variation detection. Second, the variants of SNP and InDel were detected by GATK HaplotypeCaller, then using GATK VariantFiltration to filter variant, the filtered standard as follows: a) variants with mapping qualities < 30; b) the Total Mapping Quality Zero Reads < 4; c) approximate read depth < 5; d)QUAL<50.0; e) phred-scaled p-value using Fisher's exact test to detect strand bias > 10.0. After above two steps, the data would be transformed to VCF format, variants were further annotated by ANNOVARand associated with multiple databases, such as,1000 genome, ESP6500, dbSNP , EXAC, Inhouse(MyGenostics), HGMD, and predicted by SIFT, PolyPhen-2, MutationTaster, GERP++.Variants SelectedIn this course, five steps using to select the potential pathogenic mutations in downstream analysis: (i) Mutation reads should be more than 5, mutation ration should be no less than 30%; (ii) Removing the mutation, the frequency of which showed more than 5% in 1000g, ESP6500 and Inhouse database; (iii) If the mutations existed in InNormal database (MyGenostics), then dropped; (iv) Removing the synonymous. (v) After (i),(ii),(iii), if the mutations were synonymous and they were reported in HGMD, left them. When finished above jobs, the mutations which were left should be the pathogenic mutations.