Sureselectxt human all exon v4 kit

Sufficient amounts of DNA were available for exome-sequencing from 50 JMML patients (discovery cohort and validation cohort). Sequencing libraries were prepared at the DKFZ Genomics and Proteomics Core Facility using the Agilent “SureSelectXT Human all Exon V4” kit and subsequently sequenced on a HiSeq2000 instrument using the 100 bp paired-end mode. Alignment to the hs37d5 reference genome was performed using BWA-MEM^{63 (link)}. SAMtools/BCFtools (version 0.1.19) were used for single nucleotide variants (SNV) calling^{64 (link)}. SNV found with high frequency in other mutation databases (i.e., ‘common = 1’ tag in dbSNP or > 1% frequency in ExAC 0.3.1) were filtered out and only high-confidence mutations in the coding regions were kept. Calling of small insertions/deletions (INDEL) was performed by Platypus 0.8.1 using the same filtering process as with the SNV calling^{65 (link)}. Both SNV and INDEL calling were performed without using paired germline control samples. The data were plotted as an OncoPrint using the ComplexHeatmap package^{66 (link)}. Statistics between groups were performed using the non-parametric Wilcoxon's test.

Library construction and capture hybridization was performed using the SureSelect XT Human All Exon V4 kit (Agilent Technologies) according to the manufacturer’s protocol. Samples were barcoded post-capture to allow for multiplexing of 4 samples per HiSeq2000 lane. Cluster generation took place on the Illumina cBot according to the manufacturer’s recommendations. Sequencing occurred on the Illumina HiSeq2000 using the reagents provided in the Illumina TruSeq PE Cluster Kit v3 and the TruSeq SBS Kit-HS (200 cycle) kit. An average of 81.4 million pass filter paired-end reads per sample were generated for an average depth of 45x. Filtering was performed using the software GEM.app [18 (link)].

Library preparation and sequencing of the samples in Pilot 1 and Pilot 2 were performed at BGI-Europe, Copenhagen, Denmark—and those of Pilot 3 at Broad Institute's Genomics Platform, Cambridge, USA. The total number of samples processed were in Pilot 1: 14 (7 subjects of DBS_2x3.2 and WB_ref sample types), Pilot 2: 24 (8 subjects of DBS_2x3.2, WB_ref and WB_ref replica sample types) and Pilot 3: 42 (7 subjects of DBS_2x3.2, DBS_2x1.6 (prepared in triplicate), WB_ref and WB_WGA_ref sample types). Different library preparation kits were used in the pilots. Pilot 1: SureSelectXT Human All Exon V4 kit (Agilent), Pilot 2: a BGI Capture kit (produced in-house at BGI) and Pilot 3: SureSelectXT Human All Exon V2 kit (Agilent). The libraries of Pilot 1 and Pilot 3 were prepared according to the manufacturers instructions using 1 μg of input DNA per sample. Library preparation in Pilot 2 were with 500 ng of DNA as input per sample. Library validation was with the KAPA Library Quantification Kit (KAPA Biosystems), and sequencing was performed on a HiSeq2000 Sequencer for the samples of Pilot 1 and Pilot 2 and a HiSeq2500 Sequencer for the samples of Pilot 3.