title: Feasibility of low-throughput next generation sequencing for germline DNA screening.
abstract: BACKGROUND
  Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS).
  
  
  METHODS
  Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1-12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases.
  
  
  RESULTS
  The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤ 0.15, strand biases >-35, or genotype quality scores ≤ 80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤ 5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity).
  
  
  CONCLUSIONS
  Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore;
authors.forename: Nur Sabrina
authors.initials: NS
authors.lastname: Sapari
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore;
authors.forename: Eiram
authors.initials: E
authors.lastname: Elahi
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore;
authors.forename: Mengchu
authors.initials: M
authors.lastname: Wu
authors.affiliation: Department of Epidemiology and Biostatistics, Imperial College London, London, United Kingdom;
authors.forename: Marie
authors.initials: M
authors.lastname: Loh
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore;
authors.forename: Hong Kiat
authors.initials: HK
authors.lastname: Ng
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore;
authors.forename: Xiao
authors.initials: X
authors.lastname: Han
authors.affiliation: Department of Haematology Oncology, National University Cancer Institute, National University Health System, Singapore;
authors.forename: Hui Ling
authors.initials: HL
authors.lastname: Yap
authors.affiliation: Illumina Inc., San Diego, CA;
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Klemm
authors.affiliation: Department of Pathology, National University Health System, Singapore;
authors.forename: Brendan
authors.initials: B
authors.lastname: Pang
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore;
authors.forename: Touati
authors.initials: T
authors.lastname: Benoukraf
authors.affiliation: School of Public Health, National University Health System, Singapore;
authors.forename: Yik Ying
authors.initials: YY
authors.lastname: Teo
authors.affiliation: School of Surgery, The University of Western Australia, Perth, Australia.
authors.forename: Barry
authors.initials: B
authors.lastname: Iacopetta
authors.affiliation: Department of Haematology Oncology, National University Cancer Institute, National University Health System, Singapore;
authors.forename: Soo Chin
authors.initials: SC
authors.lastname: Lee
authors.affiliation: Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Pathology, National University Health System, Singapore; richie.soong@nus.edu.sg.
authors.forename: Richie
authors.initials: R
authors.lastname: Soong
mesh_terms: D000368:Aged; D004252:DNA Mutational Analysis / Q000379:methods*; D005240:Feasibility Studies; D018095:Germ-Line Mutation / Q000235:genetics*; D006801:Humans; D008875:Middle Aged