title: Genotyping of 25 leukemia-associated genes in a single work flow by next-generation sequencing technology with low amounts of input template DNA.
abstract: BACKGROUND
  We sought to establish a convenient, sensitive next-generation sequencing (NGS) method for genotyping the 26 most commonly mutated leukemia-associated genes in a single work flow and to optimize this method for low amounts of input template DNA.
  
  
  METHODS
  We designed 184 PCR amplicons that cover all of the candidate genes. NGS was performed with genomic DNA (gDNA) from a cohort of 10 individuals with chronic myelomonocytic leukemia. The results were compared with NGS data obtained from sequencing of DNA generated by whole-genome amplification (WGA) of 20 ng template gDNA. Differences between gDNA and WGA samples in variant frequencies were determined for 2 different WGA kits.
  
  
  RESULTS
  For gDNA samples, 25 of 26 genes were successfully sequenced with a sensitivity of 5%, which was achieved by a median coverage of 492 reads (range, 308-636 reads) per amplicon. We identified 24 distinct mutations in 11 genes. With WGA samples, we reliably detected all mutations above 5% sensitivity with a median coverage of 506 reads (range, 256-653 reads) per amplicon. With all variants included in the analysis, WGA amplification by the 2 kits tested yielded differences in variant frequencies that ranged from -28.19% to +9.94% [mean (SD) difference, -0.2% (4.08%)] and from -35.03% to +18.67% [mean difference, -0.75% (5.12%)].
  
  
  CONCLUSIONS
  Our method permits simultaneous analysis of a wide range of leukemia-associated target genes in a single sequencing run. NGS can be performed after WGA of template DNA for reliable detection of variants without introducing appreciable bias.
authors.affiliation: Abteilung für Hämatologie und Internistische Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Rinke
authors.forename: Vivien
authors.initials: V
authors.lastname: Schäfer
authors.forename: Mathias
authors.initials: M
authors.lastname: Schmidt
authors.forename: Janine
authors.initials: J
authors.lastname: Ziermann
authors.forename: Alexander
authors.initials: A
authors.lastname: Kohlmann
authors.forename: Andreas
authors.initials: A
authors.lastname: Hochhaus
authors.forename: Thomas
authors.initials: T
authors.lastname: Ernst
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D004247:DNA / Q000032:analysis*; D015894:Genome, Human; D006801:Humans; D015477:Leukemia, Myelomonocytic, Chronic / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA