Interlaboratory evaluation of high molecular weight DNA extraction methods for long-read sequencing and structural variant analysis.

Background: Long-read sequencing technologies enable resolution of structural variants (SV) and long-range genome assembly, but require high molecular weight (HMW) DNA of both high quantity and quality to produce optimal sequencing results. New DNA extraction methods have been developed but these have not been assessed for use in routine testing. The interlaboratory study described here tested four commonly used methods: Fire Monkey, Nanobind, Puregene and Genomic-tip with a reference cell line containing known chromosomal alterations.

The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing.

Background: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available.

A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer's disease and brain cancers.

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer's disease (AD). However, the influence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets).

The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018).

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

Background: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use.

Control Materials and Digital PCR Methods for Evaluation of Circulating Cell-Free DNA Extractions from Plasma.

Cell-free DNA is an accessible source of genetic material found naturally in plasma that could be used in many diagnostic applications. Translation of cfDNA analysis methods from research laboratories into the clinic would benefit from controls for monitoring the efficiency of patient sample purification and for quality control of the whole workflow from extraction through to analysis. Here we describe two types of control materials that can be "spiked" into plasma samples to monitor and evaluate different aspects of the workflow.

Digital PCR dynamic range is approaching that of real-time quantitative PCR.

Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

Background: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive.

An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples.

Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR.

Digital PCR (dPCR) offers absolute quantification through the limiting dilution of template nucleic acid molecules and has the potential to offer high reproducibility. However, the robustness of dPCR has yet to be evaluated using complex genomes to compare different dPCR methods and platforms. We used DNA templates from the pathogen Mycobacterium tuberculosis to evaluate the impact of template type, master mixes, primer pairs and, crucially, extraction methods on dPCR performance.

Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis.

Background: DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS).

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses.

Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.

Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.

Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods.

Validation of high-throughput single cell analysis methodology.

High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection.

Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis.

Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level.

Applicability of RNA standards for evaluating RT-qPCR assays and platforms.

The availability of diverse RT-qPCR assay formats and technologies hinder comparability of data between platforms. Reference standards to facilitate platform evaluation and comparability are needed. We have explored using universal RNA standards for comparing the performance of a novel qPCR platform (Fluidigm® BioMark™) against the widely used ABI 7900HT system.

Validation of reference gene stability for APAP hepatotoxicity studies in different in vitro systems and identification of novel potential toxicity biomarkers.

Liver cell lines and primary hepatocytes are becoming increasingly valuable for in vitro toxicogenomic studies, with RT-qPCR enabling the analysis of gene expression profiles following exposure to potential hepatotoxicants. Supporting the accurate normalisation of RT-qPCR data requires the identification of reference genes which have stable expression during in vitro toxicology studies. Therefore, we performed a comprehensive analysis of reference gene stability in two routinely used cell types, (HepG2 cells and primary rat hepatocytes), and two in vitro culture systems, (2D monolayer and 3D scaffolds).

Evaluation of a novel approach for the measurement of RNA quality.

Background: Microarray data interpretation can be affected by sample RNA integrity. The ScreenTape Degradation Value (SDV) is a novel RNA integrity metric specific to the ScreenTape(R) platform (Lab901). To characterise the performance of the ScreenTape(R) platform for RNA analysis and determine the robustness of the SDV metric, a panel of intentionally degraded RNA samples was prepared.