title: Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates.
abstract: BACKGROUND
  Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of low-level mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:G>T:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts.
  
  
  METHODS
  We amplified selected amplicons from lung cancer FFPE DNAs using the TruSeq Cancer Panel. SNCs occurring at a frequency <10% were considered most likely to represent sequence artifacts and were enumerated for both UDG-treated and -untreated DNAs.
  
  
  RESULTS
  Massively parallel sequencing of FFPE DNA samples showed multiple SNCs, predominantly C:G>T:A changes, with a significant proportion occurring above the background sequencing error (defined as 1%). UDG pretreatment markedly reduced C:G>T:A SNCs without affecting the detection of true somatic mutations. However, C:G>T:A changes within CpG dinucleotides were often resistant to the UDG treatment as a consequence of 5-methyl cytosine being deaminated to thymine rather than uracil.
  
  
  CONCLUSIONS
  UDG pretreatment greatly facilitates the accurate discrimination of mutations in FFPE samples by use of amplicon-based approaches. This is particularly important when working with samples with low tumor purity or for the assessment of mutational heterogeneity in tumors.
authors: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.
authors: Hongdo
authors: H
authors: Do
authors: Stephen Q
authors: SQ
authors: Wong
authors: Jason
authors: J
authors: Li
authors: Alexander
authors: A
authors: Dobrovic
mesh_terms: D016477:Artifacts; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology; D018899:CpG Islands; D004247:DNA / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D009154:Mutation; D016612:Paraffin Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
chemical_list: D005557:Formaldehyde; D014498:Uracil; D004247:DNA; D051981:Uracil-DNA Glycosidase