title: Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform.
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.
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. irene.vanni@hsanmartino.it.
authors.forename: Irene
authors.initials: I
authors.lastname: Vanni
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. simona.coco@hsanmartino.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Coco
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. anna.truini@hsanmartino.it.|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. anna.truini@hsanmartino.it.
authors.forename: Anna
authors.initials: A
authors.lastname: Truini
authors.affiliation: Laboratory of Molecular Genetics, IRCCS, Giannina Gaslini Institute, L. go G. Gaslini 5, 16148 Genoa, Italy. marta.rusmini@gmail.com.
authors.forename: Marta
authors.initials: M
authors.lastname: Rusmini
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. mariagiovanna.dalbello@hsanmartino.it.
authors.forename: Maria Giovanna
authors.initials: MG
authors.lastname: Dal Bello
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. angela.alama@hsanmartino.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Alama
authors.affiliation: Laboratory of Tumor Epigenetics, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. barbara.banelli@hsanmartino.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Banelli
authors.affiliation: Department of Pathology, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. marco.mora@hsanmartino.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Mora
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. ery80x@yahoo.it.
authors.forename: Erika
authors.initials: E
authors.lastname: Rijavec
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. giulia.barletta@yahoo.it.
authors.forename: Giulia
authors.initials: G
authors.lastname: Barletta
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. carlo.genova1985@gmail.com.
authors.forename: Carlo
authors.initials: C
authors.lastname: Genova
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. febiello@gmail.com.
authors.forename: Federica
authors.initials: F
authors.lastname: Biello
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. claudia_87m@yahoo.it.
authors.forename: Claudia
authors.initials: C
authors.lastname: Maggioni
authors.affiliation: Lung Cancer Unit, IRCCS AOU San Martino-IST National Cancer Research Institute, L. go R. Benzi 10, 16132 Genoa, Italy. francesco.grossi@hsanmartino.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Grossi
mesh_terms: D000328:Adult; D000368:Aged; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D019295:Computational Biology / Q000379:methods* / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control; D012680:Sensitivity and Specificity; D057188:Workflow
keywords: FFPE; Ion Torrent PGM; NGS workflow; NSCLC; cfDNA; next-generation sequencing