Next-generation sequencing workflow for NSCLC critical samples using a targeted sequencing approach by ion torrent PGM™ platform

Irene Vanni, Simona Coco, Anna Truini, Marta Rusmini, Maria Giovanna Dal Bello, Angela Alama, Barbara Banelli, Marco Mora, Erika Rijavec, Giulia Barletta, Carlo Genova, Federica Biello, Claudia Maggioni, Francesco Grossi

Research output: Contribution to journalArticle

Abstract

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.

Original languageEnglish
Pages (from-to)28765-28782
Number of pages18
JournalInternational Journal of Molecular Sciences
Volume16
Issue number12
DOIs
Publication statusPublished - Dec 3 2015

Keywords

  • CfDNA
  • FFPE
  • Ion Torrent PGM
  • Next-generation sequencing
  • NGS workflow
  • NSCLC

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Spectroscopy
  • Inorganic Chemistry
  • Catalysis
  • Molecular Biology
  • Computer Science Applications

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  • Cite this

    Vanni, I., Coco, S., Truini, A., Rusmini, M., Dal Bello, M. G., Alama, A., Banelli, B., Mora, M., Rijavec, E., Barletta, G., Genova, C., Biello, F., Maggioni, C., & Grossi, F. (2015). Next-generation sequencing workflow for NSCLC critical samples using a targeted sequencing approach by ion torrent PGM™ platform. International Journal of Molecular Sciences, 16(12), 28765-28782. https://doi.org/10.3390/ijms161226129