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  • Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format Leonardo B. Pinheiro,*,† Helen O’Brien,‡ Julian Druce,§ Hongdo Do,∥ Pippa Kay,⊥ Marissa Daniels,#

    Jingjing You,× Daniel Burke,† Kate Griffiths,† and Kerry R. Emslie†

    †National Measurement Institute (NMI), Lindfield, Sydney, New South Wales 2070, Australia ‡Research and Development, Australian Red Cross Blood Service, Kelvin Grove, Queensland 4059, Australia §Victorian Infectious Diseases Reference Laboratory, Melbourne, Victoria 3000, Australia ∥Olivia Newton-John Cancer Research Institute, Translation Genomics and Epigenomics Laboratory, Heidelberg, Victoria 3084, Australia ⊥Agri-Bio Molecular Genetics, Biosciences Research Division, Bundoora, Victoria 3083, Australia #The Prince Charles Hospital University of Queensland, Thoracic Research Centre, Chermside, Queensland 4032, Australia ×Save Sight Institute, Sydney Eye Hospital, Sydney Medical School, University of Sydney, Sydney, New South Wales 2000, Australia

    *S Supporting Information

    ABSTRACT: Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

    Over the past few years, use of digital polymerase chainreaction (PCR) technology has rapidly expanded and diversified into a multitude of areas within life sciences. The expansion in application of digital PCR technology is evident from literature reports in various areas of research including pathogen detection,1−4 monitoring of food and water safety,5−7

    and microbial ecology.8 Because digital PCR can resolve small differences in the amount of nucleic acids, the largest uptake of this technology is in clinical research such as biomarker quantification,9−11 detection of genetic variants in cancer patients,12,13 detection of copy number alterations in stem cells,14 monitoring transplant recipients,15 quantification of massively parallel sequence libraries,16−18 and genome editing.19,20 Recognition that digital PCR can provide unprecedented levels of precision, accuracy, and resolution for quantification of nucleic acids, together with development and availability of affordable instrumentation, have been the main reasons for the rapid expansion of digital PCR.

    Transitioning digital PCR technology from a research laboratory to a clinical setting requires processes for method validation and verification and demonstration of method reproducibility and instrument performance. Many DNA reference materials have been prepared for

    validation and calibration of specific real-time quantitative PCR (qPCR) assays. Because digital PCR is a primary measurement method that does not require a calibrator and is in principle a counting technique, digital PCR has been used for property value assignment of some DNA reference materials with traceability to the International System of Units.21−25 Reference materials have also been prepared to assess biases in specific steps of a qPCR process such as biases that may arise in

    Received: December 19, 2016 Accepted: October 2, 2017 Published: October 2, 2017


    © XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b05032 Anal. Chem. XXXX, XXX, XXX−XXX

    Cite This: Anal. Chem. XXXX, XXX, XXX-XXX

  • measurement of methylated DNA after bisulphite conversion.26

    To date, such reference materials often comprise a plasmid DNA solution with an assigned copy number concentration or copy number ratio. Even if a validated PCR protocol is followed, instrument

    related factors can introduce error or bias to results. Accurate thermal cycling conditions during PCR amplification are critical for reproducible results. Inaccurate thermal cycling temper- atures or poor uniformity of temperature across heating block units can result in no amplification or low amplification efficiency in wells where optimal temperatures are not reached. PCR assays targeting guanine and cytosine (GC)-rich DNA sequences are especially prone to inefficient amplification resulting from suboptimal denaturation and annealing temper- atures.27 This was highlighted in a recent study involving a qPCR based measurement targeting a methylated, GC-rich DNA sequence in which diagnostic results were incorrectly interpreted due to instrument derived denaturation temper- ature differences between samples within 96-well plates.28

    In this study, we describe the development, characterization, and interlaboratory validation of a DNA reference material under a 96-well format designed for digital PCR instrument validation, monitoring of instrument performance, and training in digital PCR instrumentation and procedures. The reference material is produced using a “scalable” high precision robotic acoustic droplet ejection (ADE) (Labcyte) technology.29 ADE is an ultrasound-based liquid ejection technology with several advantages over traditional pipetting techniques. ADE does not require tips or pins29 for dispensing and is capable of delivering precise nanoliter volumes of solution, thus utilizing much higher DNA concentrations than required for dispensing the same amount of DNA using traditional microliter volume techniques. Together, these features reduce the risk of DNA loss due to adsorption to the surface of the pipet tip or storage tube. Unlike many other DNA reference materials, the reference value is expressed in DNA copy number amount per well and assigned by droplet digital PCR (ddPCR). DNA reference materials in this format, in addition to verifying instrument performance, could also be used to support analytical validation of specific PCR methods.

    ■ EXPERIMENTAL SECTION DNA Materials. The procedure used for production of

    DNA materials is described in Supporting Information S-1. Briefly, DNA materials were produced by end point PCR amplification of human genomic DNA (Human placental DNA, Sigma-Aldrich product number D 4642) followed by deproteination, ethanol precipitation, high-pressure liquid chromatography (HPLC), fractionation and ultrafiltration. CDKN2A_550 is a 550 base pair (bp) amplicon corresponding to part of the human cyclin-dependent kinase inhibitor 2A (CDKN2A) gene promoter region. CDKN2A_183 was prepared by digestion of CDKN2A_550 using MspI restriction enzyme (New England BioLabs) followed by the same purification procedure used for materials produced by end- point PCR amplification. TLX3_304 is a 304 bp amplicon corresponding to part of the human T-cell leukemia homeobox 3 gene promoter region. Methylated CDKN2A_550 (mCDKN2A_550 ) a nd me t h y l a t e d TLX3_304 (mTLX3_304) were prepared by in vitro methylation using M.SssI CpG Methyl transferase (New England BioLabs). NMIA NA008 Copy Number Verification Plate, High GC DNA reference material (hereafter referred to as NA008 High

    GC reference material) consists of a defined number of copies of mTLX3_304 acoustically dispensed into each well of a 96- well PCR microplate. All dilutions of DNA were performed using 1× TE0.1 buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0).

    Instrumentation. Digital PCR. QX100 and QX200 Droplet Digital PCR systems (Bio-Rad Laboratories Pty Ltd., Australia) including DG8 cartridges for droplet generators (Bio-Rad Laboratories Pty Ltd., Australia) were used by all laboratories participating in the interlaboratory study for ddPCR measure- ments, except for one laboratory (Laboratory 4) which used an automated droplet generator (AutoDG) and DG32 cartridges for AutoDG (Bio-Rad Laboratories Pty Ltd., Australia). Most Droplet Digital PCR systems included a C1000 Touch Thermal Cycler (Bio-Rad Laboratories Pty Ltd., Australia) for thermal cycling of droplets in ddPCR assays, except for three laboratories participating in the interlaboratory study; Labo- ratory 2 used an Arktik Thermal Cycler Type 5020 (Thermo Scientific) and Laboratories 5 and 6 used a T100 Thermal Cycler (Bio-Rad Laboratories Pty Ltd., Australia). For experimental work to identify DNA materials sensitive to thermal cycler temperature uniformity, Mastercycler