Bio it platform

DNA was prepared for sequencing using the TruSeq DNA PCR-free paired-end protocol (Illumina, San Diego, CA, USA) and WGS was performed on a NovaSeq 6000 instrument (Illumina) with 150 bp paired end sequencing to approximately 30X coverage with v1.5 reagents. Processing of WGS sequencing data was performed using RTA v3.4.4 and bcl2fastq v2.20.0.422, with variant calling (including both small and structural variants) on Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT platform, Ensembl Variant Effect Predictor (VEP) and AnnotSV [25 (link)]. BAM files were visualized using the Integrative Genomics Viewer (IGV) [26 (link)].

Whole genome sequencing (WGS) was done at 30X coverage for the 1653 individuals from the Gabriella Miller Kids First project, by the genomics platform of the Broad Institute. The variant call format (VCF) files of WGS were generated using the Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform (Illumina, San Diego, CA), aligned to the GRCh38/hg38 human genome assembly. For the validation dataset, WGS VCF files of participating individuals were extracted from the TOPMED database directly. The annotations for the variants were generated using the ANNOVAR software developed by our group [5 (link)], and the variants were further divided into three groups based on their genomic locations, i.e., variants in coding regions, variants in non-coding variants including intronic variants, variants in untranslated regions (UTR) or in non-coding RNAs, and variants in intergenic regions.

Raw BCL files generated by the sequencer were converted to fastq files for each sample using bcl2fastq v.2.19. Raw sequencing data are evaluated with FastQC, a tool for assessing sequencing quality. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.38. Cleaned reads were then aligned to GRCm38 reference genome using Illumina Dragen Bio-IT Platform. BAM files were generated as a result of this step.