DNA extraction
Blood:
Blood was diluted with 1 volume of DPBS (#14190, Gibbco). Sepmate tube were filled with 4ml of Lymphoprep, and diluted blood was added on top. PBMCs were separated with 10 minutes of centrifugation at 1200g. The supernatant was collected, and cells were further diluted in 10 mL of additional DPBS. After centrifugation of 8 minutes at 400g, the supernatant was discarded, and cells were resuspended in 1ml of DPBS for another 8 minutes of centrifugation at 400g. Cells were resuspended in 200 μL of DPBS with 20 μL of Proteinase K and 200 μL of Buffer AL. After vertexing, the mix was incubated for 10 minutes at 56C. 200 μL of ethanol (#459836, Sigma-Aldrich) was added to the mix and mixed by pipetting before being transferred to the DNeasy minispin column. DNA was loaded on the column by centrifugation 1 min at 6000g. DNA was purified with 500 μL of AW1 buffer and centrifuged at 6000g for 1 minute. Then washed with 500 μL of AW2 buffer then centrifuged at 20000g for 3 minutes.
Tissue samples:
Samples were thawed and resuspended in 600μL of RLT Plus buffer. Samples were homogenized with a syringe with a 20-gauge needle. The lysate was centrifuged at 20000g for 3 min and the supernatant was transferred to the AllPrep DNA spin column and centrifuged at 8000g for 1 minute. DNA was further purified by the addition of 500μL of AW1 buffer and centrifuged at 8000g for 1 minute, followed by 500μL of AW2 buffer and centrifugation at 20000g for 2 minutes.
Whole Exome Sequencing (WES)
Libraries were sequenced on Illumina’s HiSeq 4000 platform using 150 bp paired-end reads (150PE) at the Genomics Core, Sidra Medicine. Reads were mapped to reference genome hs37d5 (1000 genomes Phase2 Reference Genome Sequence) based on GRCh37/hg19 using BWA (v.0.7.12)3.
Single-nucleotide variants (SNVs) were called using mutect (v.1.1.7)4 and somatic small insertions and deletions (indels) using strelka25 (bcbio-nextgen v.1.1.1)6. To filter out false-positive SNP calls, the fpfilter7 was used, and the applied filtering parameters are specified in the fpfiler.pl script shared on GitHub. Subsequently, Mutation Annotation Format (MAF) files were generated using the VCFtoMAF tool (v.1.6.16)8 which also appended the SIFT, PolyPhen and ExAc annotations. The Personal Cancer Genome Reporter (PCGR)9 was created from the VCF files and delivered to the oncologists.