Whole-exome library preparation, exome capture and sequencing were performed using our standard protocols at the McGill University and Génome Québec Innovation Centre. For germline and fresh-frozen tumor samples, the Agilent SureSelect kits were used, following the manufacturer's protocols. FFPE tissue-derived DNA was captured using the Nextera Rapid-Capture Exome kit. The paired-end sequencing was performed using Illumina HiSeq 2000 with 100-bp length. High-quality trimmed reads were aligned to the UCSC hg19 reference genome with Burrows-Wheeler aligner (BWA) version 0.5.9 [12 (link)]. Indels were re-aligned using the Genome Analysis Toolkit (GATK) IndelRealigner [13 (link)]. Reads marked as PCR duplicates by Picard were excluded from further analysis (http://picard.sourceforge.net/ ). To ensure the best performance of MuTect and LoFreq, GATK BaseRecalibrator was used to increase the base quality score accuracy. Targeted re-sequencing was performed using a MiSeq sequencing platform with an average coverage of 5000X. Primers used in the experiments were shown in Supplementary Table 2 . Finally, a Fisher's exact test was performed to identify somatic variants from the tumor and the matched germline sample.
Sureselect kits
Manufactured by Agilent Technologies
Sourced in United States
SureSelect kits are a series of genomic enrichment products from Agilent Technologies. These kits are designed to selectively capture and enrich specific regions of the genome, enabling targeted sequencing analysis. The core function of the SureSelect kits is to facilitate the isolation and preparation of specific genomic targets for downstream sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq platform, by Illumina (2 mentions)
Hiseq 2000, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Ion ampliseq cancer hotspot panel v2, by Thermo Fisher Scientific (1 mentions)
Ion proton sequencer, by Thermo Fisher Scientific (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Qiamp dna blood midi kit, by Qiagen (1 mentions)
Fastqc, by Babraham Bioinformatics (1 mentions)
5 protocols using sureselect kits
1
Whole-Exome Sequencing of Tumor and Germline Samples
2
Whole-Exome Sequencing Workflow
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Blood, saliva, or buccal swab samples were collected from each patient, and genomic DNA was extracted from each sample. All exon regions of all human genes (~22 000) were captured using the Agilent SureSelect kits (version C2, December 2018) and sequenced using the NovaSeq platform (Illumina, San Diego, CA). The quality of FASTQ files obtained by sequencing with the Illumina Novaseq 6000 was assessed using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Subsequently, the base and sequence adapters with low base quality were removed using Trimmomatic.18 Pre‐processed FASTQ files were aligned to the reference sequence (original GRCh37 from NCBI, February 2009) by BWA‐MEM (v.0.7.17).19 Aligned BAM files were sorted and extracted using the statistical metric by samtools (v.1.9).20 Duplication was marked by Picard (v.2.20.8) (http://broadinstitute.github.io/picard/ ). Single nucleotide variants and indel variants were called by HaplotypeCaller of GATK (v.3.8).21 Finally, variant call formats (VCF) were generated. The mean depth of coverage was 100 X (>10 X = 99·2%).
3
Whole Exome Sequencing of Buccal Swab Samples
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Genomic DNA was extracted from buccal swab samples of the proband. Whole exons and their flanking intronic regions were captured using Agilent SureSelect kits (version C2, December 2018) and sequenced using the NovaSeq platform (Illumina, San Diego, CA, USA). Raw genome sequencing data were aligned to the human reference sequence GRCh37 (hg19). Differences were called and annotated. The mean depth of coverage was 100X (>10X = 99.2%). The variant interpretation was performed as described previously [13 (link),14 (link)]. Variants with minor allele frequency (MAF) < 0.005 were selected based on the population genomic databases of ExAC (http://exac.broadinstitute.org/ ), 1000 Genomes (https://www.ncbi.nlm.nih.gov/variation/tools/1000genome ), GO-ESP (http://evs.gs.washington.edu/EVS/ ), GnomAD (http://gnomad.broadinstitute.org/ ), and KRGDB consisting of 1722 Korean individuals (3444 alleles; http://coda.nih.go.kr/coda/KRGDB/index.jsp ). Splice site variants were analyzed on the basis of in silico splice predictors, such as MaxEntScan [15 (link)]. The prioritized variants were categorized based on the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guideline for the interpretation of sequence variants [16 (link)]. ExPASy was used to predict the translation product of a variant (https://web.expasy.org/translate/ ).
4
Comprehensive Genomic Profiling of Biopsied Tissues
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Targeted and whole-exome sequencing was performed using 1.0 µg of DNA extracted from biopsied tissues. Targeted genome and exome capture were performed using the Agilent SureSelect kits, NCC Oncopanel (Catalog No. 931196, Agilent) and Human All Exon V5, respectively. Sequencing was performed on the Illumina HiSeq 1500 platform using 100 bp paired-end reads (Illumina). Basic alignment and sequence quality control were performed using the Picard and Firehose pipelines. The reads were aligned against the reference human genome from UCSC human genome 19 (Hg19) using the Burrows Wheeler Aligner Multi-Vision software package. Because duplicate reads were generated during the PCR amplification process, paired-end reads that aligned to the same genomic positions were removed using SAMtools. Somatic SNVs were called by the MuTect program, which applies a Bayesian classifier to allow detection of somatic mutations with low-allele frequency. Somatic insertion/deletion mutations (indels) were called using the GATK Somatic IndelDetector (https://software.broadinstitute.org/gatk/ ). In addition, target sequencing was performed for 10 ng of DNAs using the Ion Ampliseq Cancer Hotspot Panel v2 and the Ion Proton sequencer (Thermo Fisher Scientific, Waltham, MA, USA).
5
Whole Blood DNA Sequencing and Variant Calling
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Total gDNA was extracted from whole blood for each participant using the QIAmp DNA Blood Midi Kit (Qiagen, Hilden, Germany). Strand-specific sequencing libraries for gDNAseq were prepared using 3 μg DNA and the custom-designed SureSelect kits (Agilent) according to the manufacturer's protocols. Six to seven purified libraries were multiplexed per sequence lane using 150 bp paired-end reads on a MiSeq sequencer (Illumina, San Diego, California, United States), following the manufacturer's protocols. Approximately 5M reads were generated per sample (for coverage per gene see ►Supplementary Fig. S1, available in the online version). All raw sequence reads were quality controlled using FastQC (Babraham Bioinformatics, Cambridge, United Kingdom). Reads were trimmed (removal of adaptors and indexes) with Prinseq-lite and aligned to the reference human genome (GRCh37/hg19) with Bowtie2. Genetic variants were called using GATK HaplotypeCaller v3.4.36 with the GATK Best Practice recommendations, using a minimum required quality score of ! 40.
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