Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform

International Journal of Molecular Sciences

Irene Vanni²  Simona Coco²  Anna Truini²  Marta Rusmini³  Maria Giovanna Dal Bello²  Angela Alama²  Barbara Banelli¹  Marco Mora⁴  Erika Rijavec²  Giulia Barletta²  Carlo Genova²  Federica Biello²  Claudia Maggioni²  Francesco Grossi² 

[1] Laboratory of Tumor Epigenetics, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy;Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy;Laboratory of Molecular Genetics, IRCCS, Giannina Gaslini Institute, L. go G. Gaslini 5, 16148 Genoa, Italy;Department of Pathology, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy;

关键词: next-generation sequencing;    NGS workflow;    NSCLC;    Ion Torrent PGM;    FFPE;    cfDNA;   

DOI  :  10.3390/ijms161226129

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.