Exome Sequencing of Patient-Derived Tumor Xenografts
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Corresponding Organization : Royal Marsden Hospital
Other organizations : AstraZeneca (United Kingdom), Royal Victoria Hospital, University of Ulster, Royal Marsden NHS Foundation Trust, Leicester Royal Infirmary, Royal Surrey County Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, Freeman Hospital
Protocol cited in 2 other protocols
Variable analysis
- Fragmentation of genomic DNA to 200 bp using Covaris E Series
- DNA Capture using SureSelect XT Human All Exon v4 kit (Agilent)
- Quantification of final libraries using qPCR
- Clustering of libraries at a molarity of 14.5 pmol/L
- Sequencing on Illumina HiSeq 2000 using 2 × 75 cycles of version 3 SBS chemistry
- Alignment of reads to the human reference genome (GRCh37) using Burrows-Wheeler Algorithm (v0.7.5a)
- Filtering of PCR duplicates using Picard Tools (v1.94)
- Variant calling using the GATK pipeline (v2.3.9) best practices
- Somatic change investigation using MuTect (v1.1.4) and filtering for on-target regions using bedTools (v2.17.0)
- Comparison of allele frequencies of somatic mutations between cancer and PDX samples using R (3.1.2)
- Somatic changes among germline, cancer samples, and the PDX samples
- Allele frequencies of somatic mutations in the cancer and PDX samples
- Amount of genomic DNA (30–200 ng)
- Use of human reference genome (GRCh37)
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