title: Evaluation of two highly-multiplexed custom panels for massively parallel semiconductor sequencing on paraffin DNA.
abstract: BACKGROUND
  
  
  
  OBJECTIVE
  Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.
  
  
  METHODS
  Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.
  
  
  RESULTS
  Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman's rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8-99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1-95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3-76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.
  
  
  CONCLUSIONS
  Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece; Department of Pathology, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Vassiliki
authors.initials: V
authors.lastname: Kotoula
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Aggeliki
authors.initials: A
authors.lastname: Lyberopoulou
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Papadopoulou
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Elpida
authors.initials: E
authors.lastname: Charalambous
authors.affiliation: Health Data Specialists Ltd, Athens, Greece.
authors.forename: Zoi
authors.initials: Z
authors.lastname: Alexopoulou
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Chryssa
authors.initials: C
authors.lastname: Gakou
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Sotiris
authors.initials: S
authors.lastname: Lakis
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: Eleftheria
authors.initials: E
authors.lastname: Tsolaki
authors.affiliation: Department of Haematology, "Laikon" General Hospital, University of Athens Medical School, Athens, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Lilakos
authors.affiliation: Laboratory of Molecular Oncology, Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Fountzilas
mesh_terms: D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D005557:Formaldehyde; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics*; D016612:Paraffin Embedding; D010865:Pilot Projects; D012666:Semiconductors; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D005557:Formaldehyde