Computational protocol: RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing

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Protocol publication

[…] A total of 500 ng of genomic DNA was sheared to 150–200 bp by Covaris E220 sonication (Covaris). After AMPure XP beads (Beckman Coulter) cleanup, the samples were checked for correct size distribution using 2100 Bioanalyzer system (Agilent). For manual library preparation, the fragmented genomic DNA samples were processed with end-repair, dA addition, ligation of sequencing adaptors, and two rounds of six cycles preamplification using SureSelect XT Target Enrichment System for Illumina Paired-End Sequencing library construction kit (Agilent). Next, 750 ng of amplified DNA was hybridized with a biotinylated RNA bait set (SureSelectXT Human All Exon V5; Agilent) at 65 °C for 24 hours. The captured genomic DNA fragments were enriched by DynalMyOne Streptavidin T1 beads (ThermoFisher) and amplified with barcoded index-attached primers for 12 cycles. The AMPure XP-purified libraries were checked for size distribution (300–400 bp) using Agilent Bioanalyzer and quantified using Library Quantification Kit (Kapa Biosystems). For robotic library preparation, the same conditions and procedures were applied by using Sciclone G3 NGS Work station (Perkin Elmer). A pooled library made by mixing two final libraries at equal molar ratio were clustered at 11 pM per flow cell lane using the Illumina cBot prior to sequencing on an Illumina HiSeq 2000 platform (Illumina). Sequencing reactions were run using 2 × 100 paired-end mode. Demultiplexed FASTQ files were generated with Casava v1.8.2 configureBclToFastq.pl (provided by Illumina) from the .bcl files. The multiple FASTQ files generated by this script were concatenated and primer trimmed using the ea-utilsfastq-mcf tool with the following options: “–l 30 –q 10 –u –P 33” to remove Illumina PCR and sequencing primers from the sequences. The trimmed sequences were mapped to human genome hg19 reference sequence using the Burrows-Wheeler Aligner v0.6.2 aln and sample mode in default settings. The resulting SAM files were converted to BAM format, sorted, deduplicated, and indexed using samtools and Picard. We also applied samtools to calculate the coverage as the number of total reads mapped in each defined capture regions. In addition, we also applied the principle component analysis (PCA) to investigate the coverage distribution based on the matrix of number of coverages for all 221,749 capture regions from whole genome. […]

Pipeline specifications

Software tools BaseSpace, BWA, SAMtools, Picard
Application WES analysis
Organisms Cucumber necrosis virus
Diseases Neoplasms
Chemicals Nucleotides