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

نویسندگان

  • 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
  • William Chi-shing Cho
چکیده

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.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

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

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 (F...

متن کامل

Clinical Genotyping of Non–Small Cell Lung Cancers Using Targeted Next-Generation Sequencing: Utility of Identifying Rare and Co-mutations in Oncogenic Driver Genes1

Detection of somatic mutations in non-small cell lung cancers (NSCLCs), especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM) and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gen...

متن کامل

Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here...

متن کامل

Validation of an Ion Torrent Sequencing Platform for the Detection of Gene Mutations in Biopsy Specimens from Patients with Non-Small-Cell Lung Cancer

BACKGROUND Treatment for patients with advanced non-small cell lung cancer (NSCLC) is often determined by the presence of biomarkers that predict the response to agents targeting specific molecular pathways. Demands for multiplex analysis of the genes involved in the pathogenesis of NSCLC are increasing. METHODS We validated the Ion Torrent Personal Genome Machine (PGM) system using the Ion A...

متن کامل

GLM-based optimization of NGS data analysis: A case study of Roche 454, Ion Torrent PGM and Illumina NextSeq sequencing data

BACKGROUND There are various next-generation sequencing techniques, all of them striving to replace Sanger sequencing as the gold standard. However, false positive calls of single nucleotide variants and especially indels are a widely known problem of basically all sequencing platforms. METHODS We considered three common next-generation sequencers-Roche 454, Ion Torrent PGM and Illumina NextS...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 16  شماره 

صفحات  -

تاریخ انتشار 2015