2.6. Identification of SNP and Indel Variations

Variants including SNPs and indels were called using the Genome Analysis Toolkit (GATK v3.4) pipeline, according to the Best Practice workflow. In brief, the clean reads for each sample were aligned to the human reference genome (GRCH38) using the STAR 2-pass alignment steps [31]. Then, the resulting SAM file was put through the usual Picard processing steps, including adding read group information, sorting, marking duplicates, and indexing (http://broadinstitute.github.io/picard/). Next, the RNA-Seq specific GATK tool called SplitNCigarReads was used to split the reads into exon segments and hard-clip any sequences overhanging into the intronic regions. After the indel realignment and base recalibration, we used HaplotypeCaller to call SNPs and indels, followed by variant filtering with recommended commands and parameters. We further filtered the results by removing the variants detected in less than half of the sample size. Final variants in MM patients excluded the events detected in HC. ANNOVAR was used to annotate the variants [32].