Evaluating the quantity, quality and size distribution of cell-free DNA by multiplex droplet digital PCR

Miguel Alcaide,S Rod Rassekh,Jack Hillman,Matthew Cheung,Alexander W Wyatt,David W Scott,Gerald Batist,Rebecca J Deyell,Ryan D Morin,Aly Karsan,Nathalie Johnson

doi:10.1038/s41598-020-69432-x

Abstract

Cell-free DNA (cfDNA) has become a comprehensive biomarker in the fields of non-invasive cancer detection and monitoring, organ transplantation, prenatal genetic testing and pathogen detection. While cfDNA samples can be obtained using a broad variety of approaches, there is an urgent need to standardize analytical tools aimed at assessing its basic properties. Typical methods to determine the yield and fragment size distribution of cfDNA samples are usually either blind to genomic DNA contamination or the presence of enzymatic inhibitors, which can confound and undermine downstream analyses. Here, we present a novel droplet digital PCR assay to identify suboptimal samples and aberrant cfDNA size distributions, the latter typically associated with high levels of circulating tumour DNA (ctDNA). Our assay was designed to promiscuously cross-amplify members of the human olfactory receptor (OR) gene family and includes a customizable diploid locus for the determination of absolute cfDNA concentrations. We demonstrate here the utility of our assay to estimate the yield and quality of cfDNA extracts and deduce fragment size distributions that correlate well with those inferred by capillary electrophoresis and high throughput sequencing. The assay described herein is a powerful tool to establish quality controls and stratify cfDNA samples based on presumed ctDNA levels, then facilitating the implementation of robust, cost-effective and standardized analytical workflows into clinical practice.

Highlights

Since their initial description in 1 9481, small DNA fragments travelling in the non-cellular component of internal bodily fluids and excretions have revolutionized numerous fields in public health and preventive medicine[2,3,4,5,6]
We have explored the potential of droplet digital PCR to establish a straightforward, robust and reproducible single-well assay for Cell-free DNA (cfDNA) quality controls (QC) that addresses some of the limitations exhibited by alternative methods
We have thoroughly evaluated the performance of our droplet digital PCR (ddPCR) assay in a cohort comprised by 117 plasma samples collected from cancer patients and demonstrate here its utility to evaluate the quantity, quality and fragment size distribution of cfDNA samples

Summary

Introduction

Since their initial description in 1 9481, small DNA fragments travelling in the non-cellular component of internal bodily fluids and excretions have revolutionized numerous fields in public health and preventive medicine[2,3,4,5,6]. Capillary electrophoresis allows the accurate sizing and a reasonable estimation of the absolute concentration of cfDNA s amples[21] These methods are blind to the presence of sample impurities that can undermine downstream analyses. QPCR-based approaches have been widely used to estimate the concentration of cfDNA extracts and its integrity[14,25,26] Such assays, can be negatively impacted by gDNA contamination, because it may greatly distort the ratios between short and long cfDNA-derived amplicons, and inevitably require the parallel analysis of reference samples. Our multiplex assay targets three fragment size ranges (73–165 bp; 166–253 bp; > 253 bp) from several olfactory receptor (OR) genes, together with the co-amplification of a customizable diploid locus (STAT6 in or case) for the estimation of absolute cfDNA concentrations without the need of reference samples and calibration curves. We have thoroughly evaluated the performance of our ddPCR assay in a cohort comprised by 117 plasma samples collected from cancer patients and demonstrate here its utility to evaluate the quantity, quality and fragment size distribution of cfDNA samples

Methods

Results

Conclusion