NextGenBUG 14 August 2015 Alison Meynert

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Presentation transcript:

NextGenBUG 14 August 2015 Alison Meynert Targeted BRCA1/2 sequencing of FFPE ovarian cancer tumour samples on the Ion PGM platform NextGenBUG 14 August 2015 Alison Meynert Medical Research Council Human Genetics Unit MRC Institute of Genetics and Molecular Medicine www.igmm.ac.uk

Project Ongoing collaboration with Charlie Gourley, Mike Churchman, and Robb Hollis at ECRC Archive of formalin-fixed paraffin-embedded (FFPE) ovarian cancer tumour tissue samples Patients with relapsed platinum resistant ovarian cancer treated with pegylated liposomal doxorubicin (PLD) Retrospective sequencing of BRCA1/2 to investigate the relationship of mutation status with response to treatment

Additional motivation Another project involving targeted sequencing of FFPE high grade serous ovarian cancer (HGSOC) samples in conjunction with Stratified Medicine Scotland What DNA quality metrics relate to sequence output quality? How can we filter variants that are in fact errors introduced by FFPE-induced DNA damage?

Metrics and samples DNA quality metrics RNase P Nanodrop 260/280 260/230 Qubit DIN Sequencing quality metrics: Mean read depth % reads on target Uniformity of coverage Number of low coverage targets 121 samples, 115 sequenced

0.70 -0.15 0.30 0.53 0.06 -0.20 0.38 0.68 -0.05 0.25 -0.16 -0.34 0.28 -0.03 0.13

0.12 0.26 0.42 0.06 0.14 0.21 0.10 0.27 0.25 0.13 0.14 0.03 -0.24 -0.11 -0.15 -0.08

Decision tree via regression model Criteria for success: Min 0.8 uniformity Min 60% reads on target Fail (not sequenced): 6 Fail (sequenced): 19 Success: 96 RNaseP > ~10 = 95% chance of success RNaseP < ~10 = 50/50

FFPE and DNA damage Lower yields (cross-links can cause PCR failure) Fixation artefacts C>T base substitutions from deamination Strand breaks Mutation spectra will be affected

Filtering FFPE-induced artefacts Heterozygous bi-allelic SNPs Compared mutation spectrum for BRCA1/2 in these samples to somatic SNPs from TCGA ovarian cancer exomes Selected a minimum allele frequency of 0.10

Variant summary statistics 132 variant sites across 115 samples 71 BRCA2 28 BRCA1 40 novel 11 frameshift 21 missense 1 nonsense

Acknowledgements Edinburgh Cancer Research Centre Charlie Gourley Mike Churchman Robb Hollis IGMM Bioinformatics Service Colin Semple