Sequencing reads obtained from Ion Proton were aligned to human reference sequence (human genome build-19) and variant calling was performed using Ion Torrent Variant Caller v5.0 in Torrent Suite software v5.0 (ThermoFisher Scientific). After the reads were aligned to the reference sequence, noisy and low quality reads were removed and sequence variants were detected using Ion Torrent Variant Caller Plugin software v5.0 with optimised parameters (AmpliSeq Designer, ThermoFisher Scientific) for low-frequency variant detection with minimal false-positive calls. Further, variant calls were filtered based on the technical characteristics such as (1) variant quality score, (2) variant coverage and (3) variant allele frequency. Amplicon coverage was determined via Coverage Analysis Plugin software v5.0. ThermoFisher Scientific Alignment reads and variants called, with respect to the reference human genome sequence, were viewed using Integrative Genomic Viewer software (Thorvaldsdóttir et al, 2013 (link)) and to check for strand biases, homopolymer length and sequencing errors. The variant caller plug-in generates a variant caller file or VCF file, which was subsequently imported to Ion Reporter software v5.0 (ThermoFisher Scientific) for variant annotation and filtering.