Identification of Factors Affecting the Success of Next-Generation Sequencing Testing in Solid Tumors

Am J Clin Pathol. 2016 Feb;145(2):222-37. doi: 10.1093/ajcp/aqv023. Epub 2016 Feb 12.


Objectives: Clinical laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, but there are few guidelines regarding sample quality for successful results.

Methods: We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance.

Results: Analysis of 614 clinical cases tested in 2013 using a 50-gene hotspot mutation panel identified the major cause for unsuccessful NGS analysis was DNA less than 10 ng (91%, 67/74) associated with extremely small and low cellularity samples. High success rates were associated with resection procedures (333/342, 97%) and biopsied tumor larger than 10 mm(2) (77/77, 100%). NGS can be successfully performed on bone specimens processed with formic acid-based decalcification procedures (8/11, 73%). Tumor type and paraffin block age did not affect success. We demonstrated that NGS can be carried out on samples with less than 10 ng DNA. Analysis of 408 cases tested in 2014 using an optimized workflow showed improved NGS success rates from 88% to 95% (387/408) with pronounced improvement among tiny (<10 mm(2)) samples (from 76% to 94%) as well as cytology samples (from 58% to 87%).

Conclusions: Identifying preanalytical tissue factors allows us to improve NGS performance and to successfully test tumors obtained from minimally invasive procedures.

Keywords: Ion Torrent; Next-generation sequencing; Preanalytical tissue factors; Targeted hotspot mutation; Tissue qualification.

MeSH terms

  • Biomarkers, Tumor / genetics*
  • DNA Mutational Analysis
  • DNA, Neoplasm / analysis*
  • DNA, Neoplasm / isolation & purification
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Mutation
  • Neoplasms / diagnosis*
  • Neoplasms / genetics
  • Paraffin Embedding
  • Sequence Analysis, DNA
  • Specimen Handling*
  • Tissue Fixation
  • Workflow


  • Biomarkers, Tumor
  • DNA, Neoplasm