The variability and reproducibility of whole genome sequencing technology for detecting resistance to anti-tuberculous drugs

Background: The emergence of resistance to anti-tuberculosis drugs is a serious and growing threat to public health. Next-generation sequencing is rapidly gaining traction as a diagnostic tool for investigating drug resistance in Mycobacterium tuberculosis to aid treatment decisions. However, there are few little data regarding the precision of such sequencing for assigning resistance profiles. Methods: We investigated two sequencing platforms (Illumina MiSeq, Ion Torrent PGM™) and two rapid analytic pipelines (TBProfiler, Mykrobe predictor) using a well characterised reference strain (H37Rv) and clinical isolates from patients with tuberculosis resistant to up to 13 drugs. Results were compared to phenotypic drug susceptibility testing. To assess analytical robustness individual DNA samples were subjected to repeated sequencing. Results: The MiSeq and Ion PGM systems accurately predicted drug-resistance profiles and there was high reproducibility between biological and technical sample replicates. Estimated variant error rates were low (MiSeq 1 per 77 kbp, Ion PGM 1 per 41 kbp) and genomic coverage high (MiSeq 51-fold, Ion PGM 53-fold). MiSeq provided superior coverage in GC-rich regions, which translated into incremental detection of putative genotypic drug-specific resistance, including for resistance to para-aminosalicylic acid and pyrazinamide. The TBProfiler bioinformatics pipeline was concordant with reported phenotypic susceptibility for all drugs tested except pyrazinamide and para-aminosalicylic acid, with an overall concordance of 95.3%. When using the Mykrobe predictor concordance with phenotypic testing was 73.6%. Conclusions: We have demonstrated high comparative reproducibility of two sequencing platforms, and high predictive ability of the TBProfiler mutation library and analytical pipeline, when profiling resistance to first- and second-line anti-tuberculosis drugs. However, platform-specific variability in coverage of some genome regions may have implications for predicting resistance to specific drugs. These findings may have implications for future clinical practice and thus deserve further scrutiny, set within larger studies and using updated mutation libraries

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA

Methods for applying accurate digital PCR analysis on low copy DNA samples.

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA

Characterization of AAV vectors: A review of analytical techniques and critical quality attributes

Standardized evaluation of adeno-associated virus (AAV) vector products for biotherapeutic application is essential to ensure the safety and efficacy of gene therapies. This includes analyzing the critical quality attributes of the product. However, many of the current analytical techniques used to assess these attributes have limitations, including low throughput, large sample requirements, poorly understood measurement variability, and lack of comparability between methods. To address these challenges, it is essential to establish higher-order reference methods that can be used for comparability measurements, optimization of current assays, and development of reference materials. Highly precise methods are necessary for measuring the empty/partial/full capsid ratios and the titer of AAV vectors. Additionally, it is important to develop methods for the measurement of less-established critical quality attributes, including post-translational modifications, capsid stoichiometry, and methylation profiles. By doing so, we can gain a better understanding of the influence of these attributes on the quality of the product. Moreover, quantification of impurities, such as host-cell proteins and DNA contaminants, is crucial for obtaining regulatory approval. The development and application of refined methodologies will be essential to thoroughly characterize AAV vectors by informing process development and facilitating the generation of reference materials for assay validation and calibration. [Abstract copyright: Crown Copyright © 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.

Assessment of the pre-amplification reaction on the A) linearised ADH plasmid or B) <i>Arabidopsis</i> gDNA.

<p>For each duplex assay combination, two concentrations of non pre-amplified template (High and Low) were analysed in parallel with the pre-amplified template (PA). Each sample was analysed on quadruplicate panels, with the exception of the ‘low’ non pre-amplified <i>Arabidopsis</i> gDNA that was analysed on triplicate panels, and the copy number ratio between the two Adh targets was calculated for each panel (diamond data points). Three experiments were performed (red, orange and blue diamonds) with three duplex assay combinations (Adhα-FAM:Adhβ-VIC, Adhδ-FAM:Adhβ-VIC and Adhδ-VIC:Adhβ-FAM). Horizontal black bars represent the mean ratio across all three experiments. The absolute counts used to generate this figure are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058177#pone.0058177.s008" target="_blank">Table S4</a>.</p

Assessment of molecular dropout by digital PCR.

<p>Box and whisker plot showing the effect of using different template types: <i>Arabidopsis</i> gDNA (white plots) or linearised ADH plasmid (grey plots) on molecular dropout in the data from the ‘high’ concentration template using the Adhδ-VIC:Adhβ-FAM duplex assay. The vertical axis corresponds to the number of chambers per panel in which known dropout occurred, that is, one assay (labelled with FAM or VIC) did not produce a positive signal, but the other assay did. For each data set the box plot represent the inter-quartile range with the mean, the whiskers represent the 95% range. The full range of the data set is represented by a circle.</p

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. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp® circulating nucleic acid (CNA) kit, NucleoSpin® Plasma XS (NS) kit and FitAmp™ plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-014-7835-3) contains supplementary material, which is available to authorized users

Comparison of uniplex and duplex reactions by digital PCR.

<p>(A) Graph showing the ratios calculated for the three experiments using either uniplex (grey data points) or duplex (light blue data points) reactions on the linearised ADH plasmid for two Adh ratios: Adhα-FAM:Adhβ-VIC and Adhδ-VIC:Adhβ-FAM. Each data point and its associated 95% CIs were calculated from triplicate panels on a single 12.765 dPCR array (panel-to-panel variation). The expanded uncertainty was calculated from the three experiments for each ratio using uniplex (black data points) and duplex (dark blue data points) reactions. For the uniplex reactions, the standard error of the mean for the three experiments was used to calculate the 95% CIs as the between experiment variance exceeds that of the within experiment variance. For the duplex reactions, the 95% CIs were calculated from the mean variance across the three experiments as the between and within experiment variance was very small. (B) Graph showing the ratios calculated for either linearised ADH plasmid (black diamonds) or gDNA (red diamonds) using either uniplex or duplex reactions for two Adh ratios: Adhα-FAM:Adhβ-VIC and Adhδ-VIC:Adhβ-FAM. 95% CIs were calculated from triplicate panels from a single 48.770 dPCR array. The absolute counts used to generate this figure are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058177#pone.0058177.s007" target="_blank">Table S3</a>.</p