Sureselect target enrichment system

For each sample, 3 μg of genomic DNA was sonicated into fragments, and used to construct a paired-end sequencing library with the Agilent SureSelect Target Enrichment System. Exome capture was performed with the Agilent SureSelect Human All Exon kit. Each sample was sequenced on an Illumina HiSeq2000 instrument. The sequenced reads were aligned to the human genome reference (UCSC hg 19 version) using Burrows-Wheeler Aligner (BWA). Reads qualities were recalibrated using Genome Analysis Toolkit (GATK). Picard 1.14 was used to flag duplicate reads. GATK IndelRealigner was used to realign reads around insertion/deletion sites. The Single Nucleotide Variants (SNVs) and small insertions and deletions (InDels) were generated with GATK Unified Genotyper and in parallel with the SAMtools pipeline. The called SNVs and Indels were annotated with ANNOVAR32 (link)33 (link)34 (link)35 (link)36 (link)37 (link).

A total of 100 ng (cell lines and PDX samples) or 200 ng (FFPE samples) of extracted genomic DNA (gDNA) was used as template for library construction and prepared as previously described [22 (link)]. Briefly, gDNA was fragmented to 250 base pairs using Covaris Ultrasonication (LE220 Focused-ultrasonicator, Covaris, Woburn, MA), and fragmented DNA was purified using Agencourt AMPure XP beads (Beckman Coulter, Inc. Indianapolis, IN). Size-selected DNA was then ligated to sequencing adaptors using sample-specific indices (Illumina TruSeq HT, IDT-synthesized TS-96, or IDT dual-matched indices with UMIs), libraries were constructed using Kapa HTP (Kapa Biosystems, Wilmington, MA) and quantified using qPCR (Kapa Biosystems). The SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA) was used to perform hybrid capture using either the SureSelect Human All Exon V5 bait set for whole exome enrichment, or smaller, custom-designed bait sets. For the multiplexing, index-hopping experiments, the libraries were prepared using the IDT dual-matched indexed adapters with the Kapa Hyper Prep kit (Kapa Biosystems), and the xGen AML Cancer Panel was used for capture (Integrated DNA Technologies, Inc., Coralville, IA). Sequencing platforms used (Illumina MiSeq, HiSeq 2500, HiSeq 3000/4000, or NextSeq) are listed along with the experiments (Illumina Inc., San Diego, CA).

The exome sequencing was performed by two laboratories. Of all reports, 95 reports were analysed by Genome Diagnostics Nijmegen (Nijmegen, Netherlands), and nine reports were analysed by Ambry Genetics (Aliso Viejo, CA).
For the WES performed at Genome Diagnostics Nijmegen, the targets were enriched using Agilent SureSelectXT (Agilent Technologies), and the whole-exome sequencing was performed on an Illumina HiSeq platform (BGI, Copenhagen, Denmark), followed by data processing using BWA (read alignment) and GATK (variant calling). The variants were annotated using the external laboratory’s in-house-developed pipeline. The variants were prioritised using an in-house-designed ‘variant interface’ and manual curation.^{13 (link)} For the WES performed at Ambry Genetics, the samples were prepared using a SureSelect Target Enrichment System (Agilent Technologies) or SeqCap EZ VCRome 2.0 (Roche NimbleGen) and sequenced on an Illumina HiSeq 2000 or 2500. The initial data processing, base calling, alignments and variant calls were performed using various bioinformatics tools at Ambry Genetics. The variant calls were annotated using the Ambry Variant Analyzer tool (AVA)^{26 (link)} and filtered using laboratory-devised strategies.