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Int. J. Mol. Sci. 2015, 16(12), 28765-28782; doi:10.3390/ijms161226129

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

1
Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy
2
Department of Internal Medicine and Medical Specialties (DIMI), University of Genoa, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy
3
Laboratory of Molecular Genetics, IRCCS, Giannina Gaslini Institute, L. go G. Gaslini 5, 16148 Genoa, Italy
4
Laboratory of Tumor Epigenetics, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy
5
Department of Pathology, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: William Chi-shing Cho
Received: 7 July 2015 / Revised: 18 November 2015 / Accepted: 24 November 2015 / Published: 3 December 2015
(This article belongs to the Special Issue Molecular Classification of Human Cancer: Diagnosis and Treatment)
View Full-Text   |   Download PDF [680 KB, uploaded 11 December 2015]   |  

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. View Full-Text
Keywords: next-generation sequencing; NGS workflow; NSCLC; Ion Torrent PGM; FFPE; cfDNA next-generation sequencing; NGS workflow; NSCLC; Ion Torrent PGM; FFPE; cfDNA
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

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. Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform. Int. J. Mol. Sci. 2015, 16, 28765-28782.

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