Sureselect human all exon v 2 kit

Whole-exome sequencing was performed on both affected patients. Genomic DNA was sheared with a Covaris S2 Ultrasonicator (Covaris). An adaptor-ligated library was prepared with the Paired-End Sample Prep kit V1 (Illumina). Exome capture was performed with the SureSelect Human All Exon v2 kit (Agilent Technologies), covering 38 Megabases (Mbs) of the genome. Single-end sequencing was performed on an Illumina Genome Analyzer IIx. The sequences were aligned with the human genome reference sequence (hg19/GRCh38 build), with BWA-MEM aligner [21 (link)]. Downstream processing was carried out with the Genome Analysis Toolkit (GATK) [22 (link)], SAMtools [23 (link)], and Picard Tools (http://broadinstitute.github.io/picard/). Substitution and indel calls were both made with GATK HaplotypeCaller v3.3. All calls with a Phred-scaled quality ≤30 were filtered out. Variant annotation was based on the Human genome assembly GRCh38 as implemented in the Ensembl browser (release 88) as previously described [24 (link)–26 (link)].

Libraries were prepared with the SureSelect Human All exon V2 kit (Agilent Technologies Inc, USA). Paired-end sequencing was performed on the Illumina Genome Analyzer II platform (Illumina Inc, USA). The sequence reads were aligned to the Human Reference Genome Build hg19 using the BWA software^{18 (link)}, followed by GATK base quality score recalibration, duplicate marking and local realignment. SNPs and indels were simultaneously called in all samples using the GATK HaplotypeCaller algorithm, applying hard-filtering parameters according to GATK best practices recommendations¹⁹ . Variants were annotated and prioritized using ediva, our in-house pipeline (www.ediva.crg.eu), which provides information on minor allele frequencies from various databases, including the Exome Aggregation Consortium, and Exome Variant Server-NHBLI, variant functional effect prediction scores by SIFT, Polyphen2, MutationAssessor and CADD, and variant conservation scores PhyloP and Gerp++. Fastq files can be accessed through EGA (EGAS00001003510).

Targeted-capture sequencing was performed using a Roche NimbleGen custom 385K capture array. Paired-end sequencing libraries were generated after capture and sequenced on an Illumina sequencer. Whole-exome sequencing libraries were prepared using the Sure-Select Human All Exon v2 kit (Agilent) and sequenced on an Illumina sequencer. Whole-genome sequencing was performed by Complete Genomics. Targeted-capture and whole-exome data were analyzed using GATK (Van der Auwera et al. 2013 (link)). Whole-genome sequencing data were analyzed using Complete Genomics software. Variants were annotated with ANNOVAR (Wang et al. 2010 (link)) and filtered if their allele frequency was ≥1% in any of the following public variant databases: ExAC (Lek et al. 2016 (link)), 1000 Genomes Project (The 1000 Genomes Project Consortium 2015 (link)), NHLBI Exome Sequencing Project (Tennessen et al. 2012 (link)), and Complete Genomics 69 control genomes (Drmanac et al. 2010 (link)). See Supplemental Methods for further details.

WES was performed from cell pellets, matched-tumour tissue and germline DNA extracted from blood at the Broad Institute Genomics Platform using the Illumina HiSeq 2000 platform. The Agilent SureSelect Human All Exon v2 kit was used for capture and paired end 76 bp reads were obtained. This resulted in a mean target coverage of 102 × for the tumour and 57 × for CLF-PED-015-T. We filtered out common single nucleotide polymorphism (SNPs) that were validated in dbSNP and likely artifacts by setting minimum coverage and allele frequency thresholds.