Agilent sureselect clinical research exome v2

Ten microlitres of each normalised DNA library were pooled and incubated at 96 °C for 2 min. The library pool solution was mixed, centrifuged briefly and incubated on ice for 5 min. Libraries were sequenced on an Illumina Nova-Seq instrument. DNA libraries were prepared using the hybrid capture-based TruSight Oncology 500 (TSO500) Library Preparation Kit (Illumina, San Diego, CA, USA) following Illumina’s TSO500 reference guide targeting 523 cancer-relevant genes with a target capture size of 1.94 megabases (Mb) (see supplementary data 2 for a list of genes). The panel includes the following Meningioma-related genes and targets from the literature: NF2, KDM5C, KDM6A, SMARCB1, AKT1, mTOR, SMO, TRAF7, KLF4, PIK3CA, BAP1, TP53, CDKN2A, CDKN2B, TERT (+ promoter), NAB2-STAT6 fusion. Mean sequencing yield in the TSO500 data was 18 gigabases (Gb) per sample, with a mean unfiltered depth of coverage of 9278 reads per sample.
A subset of 13 tumours was also sequenced using the Agilent SureSelect Clinical Research Exome V2 (Agilent, Santa Clara, CA, USA) with a capture size of 67.3 Mb. Mean sequencing yield in the exome data was 46 Gb per sample, with a mean unfiltered depth of coverage of 688 reads per sample.

Library preparation and capture for exome sequencing was performed at the Kinghorn Centre for Clinical Genomics at the Garvan Institute of Medical Research using Agilent SureSelect Clinical Research Exome v2 (Agilent). Sequencing was performed on a HiSeqX (Illumina) to an average depth of coverage of 100× across captured regions. Alignments and variant calls were generated using SoftGenetics NextGene (version 2.4.1, SoftGenetics) to the February 2009 human genome assembly (GRCh37/hg19), and variant calls were restricted to coding regions and the canonical splice sites of an epileptic encephalopathy gene panel. Variants identified were then annotated using Alamut Batch (version 1.9, Interactive Biosoftware) and classified according to the American College of Medical Genetics (ACMG) criteria (Richards et al. 2015 (link)).

DNA isolation from peripheral blood of patient and parents was performed using the Chemagic DNA Blood 4 k Kit (PerkinElmer, Waltham, MA, United States). Three micrograms of double-stranded DNA were fragmented (Covaris, Woburn, MA, United States) and exonic sequences were captured using the Agilent SureSelect Clinical Research Exome V2 (Agilent, Santa Clara, CA, United States). Paired-end sequencing was performed on a HiSeq 4,000 platform (150bp paired end) and an average coverage of at least 50X. Reads were mapped against the human reference genome GRCh37/hg19 with the Burrows-Wheeler Aligner (Li and Durbin, 2009 (link)), and variants were called using the Genome Analysis toolkit (Broad Institute, Cambridge, MA, United States). Alissa Interpret software (Agilent, Santa Clara, CA, United States) was used to filter and prioritize variants. Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for the interpretation of sequence variants (Richards et al., 2015 (link)).