Targeted Exome Sequencing Protocol

The coding exons plus UTRs were captured with SureSelect All Exon v4 (Agilent Technologies) as described previously (Rohland and Reich 2012 (link)), with a few modifications. The DNA was sheared into fragments of ∼175 bp using the Covaris system (Covaris). The sheared DNA was purified with Agencourt AMPure XP SPRI beads (Beckman Coulter). The DNA was blunted with 5′-phosphorylated ends using the NEB Quick Blunting Kit and ligated to truncated PE P5 adaptors and barcoded P7 adaptors using the NEBNext Quick Ligation Module. After clean-up with Agencourt AMPure XP SPRI beads and nick fill-in with the Bst DNA polymerase, Large Fragment (New England BioLabs), the DNA fragments with adaptors were enriched by PCR. A total of 500 ng of DNA pooled from four barcoded libraries were used for hybridization and post-hybridization amplification following the manufacturer's protocol (SureSelect^XT Target Enrichment System for Illumina Paired-End Sequencing Library, Version 1.3.1, Agilent Technologies). The post-hybridization amplification product was quality checked and sequenced on an Illumina HiSeq 2500 (read lengths of 2 × 100 bp).