Library preparation and sequencing
Genomic libraries of whole-exome were constructed using 1 ug of genomic
DNA and the Sureselect QXT V6 (Agilent Technologies - Patients 1 and 2,
and Patient 1’s mother), OneSeq Constitutional Research Panel (Agilent
Technologies - Patient 2’s father), and xGen Exome Research Panel v1.0
(IDT - Integrated DNA Technologies - FFPE tumor sample) kits. Enriched
libraries were sequenced on the Illumina HiSeq 2500 platform in paired
end reads. The sequences were aligned to the GRCh37/hg19 human genome
reference with the BWA_MEM algorithm 30. Picard tools
(v.1.8, http://broadinstitute.github.io/picard/) were used to convert
the SAM file into BAM and to mark PCR duplicates. The Genome Analysis
Toolkit (GATK 3.7) 31 were used to realign indels,
recalibrate the bases, and to call (Unified Genotyper) and recalibrate
variants (VQSR). Finally, multiallelic variants were split into
different lines using the script split_multiallelic_rows.rb from
Atlas2 32 to obtain the VCF files used for analysis.