Library preparation and whole-exome sequencing (WES)
DNA samples were extracted by phenol-chloroform followed by ethanol
precipitation [27]. Genomic libraries were constructed with 1 µg of
genomic DNA using the following kits: Sureselect QXT V6 (Agilent
Technologies), OneSeq Constitutional Research Panel (Agilent
Technologies), or xGen Exome Research Panel v1.0 (IDT - Integrated DNA
Technologies). The sequencing of enriched libraries was performed on the
Illumina HiSeq 2500 platform using 150 base paired-end reads. The
sequences were aligned to the GRCh37/hg19 human genome reference with
the BWA_MEM algorithm [28]. 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) [29] was used to realign indels, recalibrate the bases,
and call (Unified Genotyper) and recalibrate variants (VQSR). Finally,
multiallelic variants were split into different lines using the script
split_multiallelic_rows.rb from Atlas2 [30] to obtain the VCF
files used for analysis.