title: Clinical Evaluation of a Novel Nine-Gene Panel for Ion Torrent  PGM Sequencing of Myeloid Malignancies.
abstract: OBJECTIVE
  In the last decade, a number of genes have been reported to be recurrently associated with myeloid malignancies. While some mutations are easily detectable by conventional molecular genetics methods, other mutations are more difficult to screen because of lower frequency and being scattered along large genomic ranges. However, newly developed approaches for next-generation sequencing provide an affordable solution for targeted multiplex resequencing of up to several hundreds of amplicons. Here, we aimed to develop and validate a novel custom panel for targeted resequencing of myeloid malignancy samples using the Ion PGM(™) System (Ion Torrent, Paisley, UK).
  
  
  METHODS
  We designed a pool of 424 primers for the amplification of 212 amplicons covering 99.46 % of the exonic regions of nine human genes as follows: ASXL1, EZH2, CALR, RUNX1, SETBP1, SF3B1, SRSF2, TET2, and U2AF1. Initial testing of the panel performance was performed on an Ion PGM(™) machine using PGM(™) 316 v2 chips on 16 DNA samples from patients with myeloid malignancies. Sequence alignment, variant calling, and annotation were performed using Ion Reporter software.
  
  
  RESULTS
  We identified a total of 14 nonsynonymous somatic coding variants in seven samples affecting six of the genes in the panel (ASXL1, CALR, RUNX1, SRSF2, TET2, and U2AF1). Notably, three of the identified mutations were not present in the Cosmic v.67 release.
  
  
  CONCLUSIONS
  This proof-of-concept study confirms the feasibility of Ion Torrent systems for resequencing of clinically relevant mutations in myeloid malignancies. It can be particularly useful in cases without the most frequent clonal markers.
authors.forename: Milena
authors.initials: M
authors.lastname: Ivanova
authors.forename: Velizar
authors.initials: V
authors.lastname: Shivarov
authors.forename: Ivan
authors.initials: I
authors.lastname: Pavlov
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Lilakos
authors.forename: Elissaveta
authors.initials: E
authors.lastname: Naumova
mesh_terms: D002352:Carrier Proteins / Q000235:genetics; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D049308:Dioxygenases; D000071221:Enhancer of Zeste Homolog 2 Protein; D005091:Exons; D056726:Genetic Association Studies; D015894:Genome, Human; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000175:diagnosis* / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics; D063151:Polycomb Repressive Complex 2 / Q000235:genetics; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics; D000072260:RNA Splicing Factors; D012097:Repressor Proteins / Q000235:genetics; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D000068103:Serine-Arginine Splicing Factors; D000072668:Splicing Factor U2AF
chemical_list: C473949:ASXL1 protein, human; D002352:Carrier Proteins; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D000072260:RNA Splicing Factors; C493728:RUNX1 protein, human; D012097:Repressor Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D012261:Ribonucleoproteins; C425066:SETBP1 protein, human; C112491:SF3B1 protein, human; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D049308:Dioxygenases; C541081:TET2 protein, human; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2