Sureselectxt target enrichment system for paired end sequencing library version 1

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).

The genomic DNA of primary cells was sequenced by next generation sequencing (NGS) to uncover the exome mutations of KRAS and TP53. A total of 150 ng to 1 µg of DNA extracted from primary cells by MALBAC (multiple annealing and looping-based amplification cycles) was sheared into fragments around 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 P7 adaptors and barcoded P5 adaptors using NEBNext Quick Ligation Module. After cleanup with Agencourt AMPure XP SPRI beads and nick fill-in with Bst polymerase large fragment (New England Biolabs), the DNA fragments with adaptors were enriched by PCR. A total amount of 500 ng of DNA pooled from four barcoded libraries was used for hybridization and posthybridization amplification following the manufacturer's protocol (SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library, version 1.3.1, February 2012, pp37-60; Agilent Technologies). The posthybridization amplification product was quality checked and sequenced with Illumina HiSEq. 2000/2500 2X 100-bp paired-end (PE) reads.