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Title: Development of a pipeline and protocols for next generation sequencing of blood and formalin-fixed, paraffin-embedded tumour DNA samples
Author: Naven, Marc
ISNI:       0000 0004 5915 0062
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2015
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Using existing software and six novel scripts, we developed a pipeline for variant calling using exome re-sequencing data of germline blood deoxyribonucleic acid (DNA) samples. We observed >99% (6,723/6,739) concordance between calls from our pipeline and previous genotyping. We identified >93% of single nucleotide variants (SNVs) and >94% of insertion/deletions called by a commercial partner using the same sequencing reads. Using a subset of genes, we showed that around half of predicted pathogenic variants could be validated in vitro. Together, these data showed that our pipeline was reliable for variant calling. Next generation sequencing (NGS) of DNA from formalin-fixed, paraffin-embedded (FFPE) tumours is technically challenging. We sought to determine the sensitivity of NGS to detect known somatic hotspot mutations (n=25) in 19 FFPE advanced colorectal cancers and to optimise it for the identification of novel somatic variants. Analysis using Illumina’s MiSeq software identified 100% of somatic mutations with 93% specificity, whereas the Genome Analysis Tool Kit’s (GATK) HaplotypeCaller identified 80-92% of somatic mutations with 100% specificity. We investigated the background mutator profile and found that normal FFPE DNA had 3-fold more SNVs than matched blood DNA, with an excess of FFPE-associated C:G>T:A substitutions (27.1 vs. 5.3%). Uracil DNA glycosylase treatment reduced this excess. Only ~5% of variants were consistently called in replicate runs and were likely to be genuine somatic variants. We detected potential candidates for genetic biomarkers of cetuximab-resistance in colorectal cancers that were previously shown to be wild type for KRAS, NRAS, BRAF and PIK3CA. We found that 7/21 (33%) of the CRCs analysed harboured mutations at either codon 12 or 61, whileother CRCs carried truncating KRAS mutations. NRAS also carried a codon 12 mutation in 1 CRC, and PIK3CA possessed mutations at codons 542 and 545. PTEN was found to harbour 5 truncating mutations at codons 71, 140, 155, 178 and 189, potentially leading to loss of function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: RC0254 Neoplasms. Tumors. Oncology (including Cancer)