Real time analysis software sequence pipeline

Quality control of sequencing data was performed in all samples using the Real-Time Analysis software sequence pipeline from Illumina. The short-read sequences were aligned against the human reference genome (Build Hs37d5, based on NCBI GRCh37) using the Burrows–Wheeler aligner (BWA-MEM) algorithm. Subsequent mutational analysis was performed at mean coverage depth ≥200 reads. Variants were filtered out when the alternative allele depth was lower than 10 reads. The GATK Mutect2 toolkit (https://gatk.broadinstitute.org/) was used for single nucleotide variant (SNV) calling. Variant annotation was performed using several resources and databases such as: SnpEff, dbNSFP, PhyloP, SIFT, PolyPhen2, MutationTaster, LRT, and CADD. The GnomAD resource (https://gnomad.broadinstitute.org/) was used to evaluate variant frequency in the global population. All mutations were evaluated using the Integrative Genomics Viewer (https://software.broadinstitute.org/software/igv/). To perform a comparative analysis of the mutational profile identified in our cohort of patients (HBU), we analyzed rectal cancer datasets obtained from The Cancer Genome Atlas (TCGA) and the Memorial Sloan Kettering Cancer Center project (MSKCC) retrieved from the cBioPortal resource (http://www.cbioportal.org/). The DNA sequencing data can be found at SRA (ID: PRJNA633284) and Supplementary Data 1.

Germline WES data were available from previous studies [12 (link),21 (link),31 (link)]. WES was performed in tumor samples of selected patients using the HiSeq2000 platform (Illumina, San Diego, CA, USA) and SureSelectXT Human All Exon v5 kit (Agilent, Santa Clara, CA, USA) for exon enrichment. Indexed libraries were pooled and massively parallel-sequenced using a paired-end 2 × 75 bp read length protocol.
The quality control of sequencing data was made in all samples previous to their analysis using the Real-Time Analysis software sequence pipeline (Illumina). Additionally, the proportion of all shared exome regions sequenced with a coverage ≥ 10× was evaluated for tumor samples. A good ratio of shared regions with high coverage (≥ 70%) was expected in good-quality samples, whereas low-quality ones were characterized by a significant drop in this percentage.
WES data analysis was performed in accordance with the workflow displayed in Figure 2. The Burrows–Wheeler Aligner (BWA-MEM algorithm) was used for read mapping to the human reference genome (build hs37d5, based on NCBI GRCh37) [43 (link)]. PCR duplicates were discarded using the MarkDuplicates tool from Picard, and then indel realignment and base quality score recalibration were performed with the Genome Analysis Toolkit (GATK, Broad Institute, Cambridge, USA) [44 (link)].