title: Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.
abstract: The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM), and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE). Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH) analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used to increase power and compensate for increased error rates.
authors.affiliation: Department of Oral Pathology, King's College London Dental Institute, London, United Kingdom. angela.stokes@kcl.ac.uk
authors.forename: Angela
authors.initials: A
authors.lastname: Stokes
authors.forename: Ignat
authors.initials: I
authors.lastname: Drozdov
authors.forename: Eliete
authors.initials: E
authors.lastname: Guerra
authors.forename: Christos A
authors.initials: CA
authors.lastname: Ouzounis
authors.forename: Saman
authors.initials: S
authors.lastname: Warnakulasuriya
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gleeson
authors.forename: Mark
authors.initials: M
authors.lastname: McGurk
authors.forename: Mahvash
authors.initials: M
authors.lastname: Tavassoli
authors.forename: Edward W
authors.initials: EW
authors.lastname: Odell
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics; D056915:DNA Copy Number Variations; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D005557:Formaldehyde; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D007834:Lasers; D019656:Loss of Heterozygosity; D046228:Microarray Analysis / Q000379:methods; D042282:Microdissection / Q000379:methods; D009059:Mouth Diseases / Q000235:genetics; D009132:Muscles / Q000378:metabolism; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde; D004247:DNA