Defining Mutations to Track

For the HapMap panels, VCF files were taken from the Genome in a Bottle Consortium⁴⁹ (NA12878) and 1000 Genomes project⁵⁰ (NA19238). Sites specific to NA12878 were subsampled to create MAF files and were subsequently run through probe design to create the 438 and 10,000 SNV (single nucleotide variant) fingerprints.

Tumor DNA was extracted from fresh-frozen tumor samples. All patients’ tumor DNA underwent whole-exome sequencing to identify trackable mutations for conventional capture. Of the four patients selected for MAESTRO, tumor DNA underwent PCR-free whole-genome sequencing. Illumina output from whole-genome sequencing was processed by the Broad Picard pipeline and aligned to hg19 using BWA. We ran the GATK best practices workflow on the Terra platform to detect somatic SNVs and indels in our deep whole-genome sequencing data using tumor/normal calling (see Terra workflow). We subset the somatic mutation calls to only SNVs and passed the candidate SNVs for tracking to our probe design pipeline. By sequencing each patient’s tumor and normal to adequate depth we can avoid tracking variants arising from clonal hematopoiesis.