Variant studio v2

To identify a potential candidate gene, genomic DNA of one of the affected individuals (V-4) was used to perform whole exome sequencing. The sample was prepared using Agilent SureSelect Target Enrichment Kit by following the manufacturer’s guide. The libraries were sequenced with Illumina HiSeq 2000/2500 sequencer. The BWA Enrichment application of BaseSpace (Illumina Inc. 5200 Illumina Way|San Diego, CA, 92122, USA) was used to analyze the reads. Alignment was performed with Burrows-Wheeler Aligner (BWA) and the variants were called with Genome Analysis Toolkit (GATK). All the called variants were annotated with Illumina Variant Studio v2.2. Based upon the inheritance pattern observed in the pedigree of the present family, we filtered the biallelic variants. Exome sequence data was carefully analyzed and the presence of all suspected homozygous variants was checked in the public databases [dbSNP (https://www.ncbi.nlm.nih.gov/snp/ accessed on 10 February 2022), 1000 Genomes browser (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/ accessed on 10 February 2022), Exome Variant Server (https://evs.gs.washington.edu/EVS/ accessed on 10 February 2022), gnomAD (https://gnomad.broadinstitute.org/ accessed on 10 February 2022)], and the exome data of 53 healthy controls collected from different ethnic groups of Pakistan.

Library preparation, indexing and target enrichment for exome sequencing was carried out using the Nextera Rapid Capture Exome kit (Illumina), targeting 45 Mb of exonic coding sequence. Paired-end (2 × 87 cycle) single-plex sequencing was carried out on a MiSeq instrument (Illumina). Secondary data analysis, including alignment of reads against human reference genome hg19, was carried out using MiSeq Reporter v2.5.1 (Illumina) and variants were called and annotated using Illumina Variant Studio v2.2. Homozygous or compound heterozygous variants with allele frequency <1%, predicted to result in loss of protein function, were considered as potential causes of the MAM-negative phenotype. Selected alignments were further visualised using Integrative Genomics Viewer (IGV v2.3)^{28 (link),29 (link)}.