Next-Generation Sequencing Analysis for Rare Variants in Type 1 Diabetes: Sample nPOD-6180
The genomic DNA sample was randomly fragmented by sonication (Covaris) to the size of 180-280 bp fragments. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. After adenylation of 3' ends of DNA fragments, adapter oligonucleotides were ligated. DNA fragments with ligated adapter molecules on both ends were selectively enriched in a PCR reaction. After PCR reaction, the libraries were hybridized in buffer with biotin-labeled probes, then magnetic beads with streptomycin were used to capture the exons. After washing beads and digesting probes, the captured libraries were enriched in a PCR reaction to add index tags. Products were purified with AMPure XP system (Beckman Coulter) and quantified by using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system. Captured DNA libraries were sequenced with the Illumina Novaseq 6000. Burrows-Wheeler Aligner (BWA) was utilized to map the paired-end clean reads to the GRCh37/hg19 human reference genome (PMID:19451168).  GATK was used for SNP/InDel detection (PMID: 25431634). ANNOVAR was used for variant annotation (PMID:20601685). Other variant annotations include ACMG Classification (PMID:25741868), SIFT Function Prediction (PMID:12824425), PolyPhen-2 Function Prediction (PMID:23315928), CADD Score (PMID: 24487276), gnomAD Frequency (PMID: 32461654), HGMD (PMID: 32596782), CLINVAR (PMID: 29165669) and CentoMD (PMID:28116331).