Study of Preanalytic and Analytic Variables for Clinical Next-Generation Sequencing of Circulating Cell-Free Nucleic Acid

The Journal of Molecular Diagnostics : JMD
Meenakshi MehrotraRajyalakshmi Luthra

Abstract

Detection of mutations in plasma circulating cell-free DNA (cfDNA) by next-generation sequencing (NGS) has opened up new possibilities for monitoring treatment response and disease progression in patients with solid tumors. However, implementation of cfDNA genotyping in diagnostic laboratories requires systematic assessment of preanalytical parameters and analytical performance of NGS platforms. We assessed the effects of peripheral blood collection tube and plasma separation time on cfDNA yield and integrity and performance of the Ion PGM, Proton, and MiSeq NGS platforms. cfDNA from 31 patients with diverse advanced cancers and known tumor mutation status was deep sequenced using targeted hotspot panels. Forty-five of 52 expected mutations and two additional mutations (KRAS p.Q61H and EZH2 p.Y646F) were detected in plasma through a custom bioinformatics pipeline. We observed comparable cfDNA concentration/integrity between collection tubes within 16 hours of plasma separation and equal analytical performance among NGS platforms, with 1% detection sensitivity for cfDNA genotyping.

Citations

Sep 28, 2017·Journal of Clinical Laboratory Analysis·Sana SahengbiekeJingjing Wu
Feb 22, 2018·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Sinchita Roy-ChowdhuriRajyalakshmi Luthra
Oct 14, 2018·Modern Pathology : an Official Journal of the United States and Canadian Academy of Pathology, Inc·Ana M BolivarRussell R Broaddus
Feb 1, 2019·Current Gastroenterology Reports·David G MenterScott Kopetz
Dec 16, 2019·Molecular Diagnosis & Therapy·Laure SorberPatrick Pauwels
May 12, 2018·Diagnostics·Francesc Castro-GinerNicola Aceto
Jun 5, 2020·The Journal of Molecular Diagnostics : JMD·Teresa GerberPeter F Ambros
Mar 9, 2021·Expert Review of Molecular Diagnostics·Daniel AnderssonAnders Ståhlberg
Jul 6, 2021·Molecular Diagnosis & Therapy·Giuseppa De Luca, Mariella Dono
Feb 29, 2020·Transfusion Medicine and Hemotherapy : Offizielles Organ Der Deutschen Gesellschaft Fur̈ Transfusionsmedizin Und Immunham̈atologie·Sandra Wienzek-LischkaGregor Bein
Aug 12, 2019·The Journal of Molecular Diagnostics : JMD·Saradhi MallampatiRajyalakshmi Luthra

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