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DNA-Based Sequencing Assays

  • Pasquale Pisapia
  • Miriam Cieri
  • Francesco Pepe
  • Umberto Malapelle
  • Giancarlo Troncone
Chapter

Abstract

Modern cytopathologists need to incorporate molecular testing into their clinical practice and be able to translate molecular assays into patient care. The detection of genomic alterations may not only refine uncertain morphological diagnoses but may also provide crucial theranostic information. Gene sequencing remains a core technology for molecular cytopathology. Sanger sequencing has paved the way for the development and the clinical implementation of next-generation sequencing (NGS) assays. In this chapter, the NGS workflow is described, focusing mainly on the technical aspects of NGS analysis, the diverse platforms available, the principles underlying the selection of targeted gene panels, and the issues related to validation of the “wet” procedure and the bioinformatics pipeline.

Keywords

NGS Molecular cytopathology Personalized medicine Gene panel Validation Cytologic samples Oncogene Sequencing by synthesis Molecular analysis 

Abbreviations

BRAF

v-raf murine sarcoma viral oncogene homolog B

DNA

Deoxyribonucleic acid

EGFR

Epidermal growth factor receptor

IVD

In vitro diagnostic

KIT

KIT proto-oncogene receptor tyrosine kinase

KRAS

Kirsten rat sarcoma viral oncogene homolog

LDT

Laboratory developed tests

LOD

Limit of detection

NGS

Next-generation sequencing

NRAS

Neuroblastoma RAS viral oncogene

PDGFRA

Platelet-derived growth factor receptor A

PPA

Positive percentage agreement

PPV

Positive predictive value

RT-PCR

Real-time polymerase chain reaction

TAT

Turnaround time

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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Pasquale Pisapia
    • 1
  • Miriam Cieri
    • 1
  • Francesco Pepe
    • 1
  • Umberto Malapelle
    • 1
  • Giancarlo Troncone
    • 1
  1. 1.Department of Public HealthUniversity of Naples “Federico II”NaplesItaly

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