Targeted enriched libraries were prepared using the SureSelectXT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced with a paired-end protocol on a HiSeq 3000 Sequencer (Illumina, Little Chesterford, UK). The quality control of the raw sequence data, base quality scores, GC content and duplications were checked using java based FastQC software (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Sequence adaptors were removed with Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ ). Sequences were then aligned against the reference genome (hg19/GRCh37) using the Burrows–Wheeler Aligner BWA (v0.7.12-r1.39) [27 (link)]. SAM files were converted to BAM files with SAMtools then sorted by Picard tools (v2.5.0) (https://broadinstitute.github.io/picard/ ), which were also used to remove PCR duplicates. BAM files were realigned locally around the indels using the Genome Analysis Tool Kit GATK (https://gatk.broadinstitute.org/hc/en-us ) (v3.5) [28 (link), 29 (link)]. The GATK HaplotypeCaller function was used to call small indels and single nucleotide variants (SNVs) in genomic variant call format (g.VCF). The variant list was then annotated using Variant Effect Predictor (VEP) software [30 (link)].
Protocol detail
Targeted Exome Sequencing Data Analysis
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Exon
Genomic
Human
Indels
Nucleotide
Corresponding Organization :
Other organizations : St James's University Hospital, University of Leeds, Wellcome Trust, St Mary's Hospital, Benha University
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Variable analysis
independent variables
- Targeted enriched libraries were prepared using the SureSelectXT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA)
- Sequences were then aligned against the reference genome (hg19/GRCh37) using the Burrows–Wheeler Aligner BWA (v0.7.12-r1.39)
- The GATK HaplotypeCaller function was used to call small indels and single nucleotide variants (SNVs) in genomic variant call format (g.VCF)
- Sequenced with a paired-end protocol on a HiSeq 3000 Sequencer (Illumina, Little Chesterford, UK)
- The quality control of the raw sequence data, base quality scores, GC content and duplications were checked using java based FastQC software
- Sequence adaptors were removed with Trim Galore
- BAM files were realigned locally around the indels using the Genome Analysis Tool Kit GATK (v3.5)
- The variant list was then annotated using Variant Effect Predictor (VEP) software
- No explicit control variables mentioned
- No positive or negative controls specified
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