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.