J Clin Pathol doi:10.1136/jclinpath-2013-201549
  • Short report

Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues

  1. Sabine Merkelbach-Bruse2
  1. 1Cologne Center for Genomics, University of Cologne, Cologne, Germany
  2. 2Institute of Pathology, University Hospital Cologne, Cologne, Germany
  3. 3Gerhard-Domagk-Institute of Pathology, University Hospital Muenster, Muenster, Germany
  1. Correspondence to Dr Margarete Odenthal, Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, Cologne 50924, Germany; m.odenthal{at}
  • Received 16 February 2013
  • Revised 20 March 2013
  • Accepted 21 March 2013
  • Published Online First 25 April 2013


Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a set of therapy relevant gene loci. DNA from macrodissected tumour samples of formalin fixed biopsies was used for PCR amplification of EGFR exons 18, 19, 21 and KRAS exon 1. A total of 128 PCR products were analysed by conventional termination sequencing as well as by ISS. Sensitivity of ISS was additionally determined using 100–10 000 copies of reference mutants. All somatic mutations detected by direct Sanger sequencing were also identified by ISS. No additional mutants were detected. Running samples with limited copies of mutated alleles revealed high sensitivity, detecting less than 10% (2500 copies) mutants in a human wild type background. In conclusion, multiplexed mutation analyses by ISS is an efficient technology that can easily be linked to existing PCR approaches in molecular pathology.