Publications by authors named "Simon Cowen"

22 Publications

  • Page 1 of 1

Pushing the Envelope with Clinical Use of Digital PCR.

Clin Chem 2021 Jul;67(7):921-923

Molecular & Cell Biology, National Measurement Laboratory, LGC, Teddington, UK.

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http://dx.doi.org/10.1093/clinchem/hvab082DOI Listing
July 2021

Evaluation of a Common Internal Standard Material to Reduce Inter-Laboratory Variation and Ensure the Quality, Safety and Efficacy of Expanded Newborn Screening Results When Using Flow Injection Analysis Tandem Mass Spectrometry with Internal Calibration.

Int J Neonatal Screen 2020 Nov 19;6(4). Epub 2020 Nov 19.

Department of Medical Biochemistry, Immunology & Toxicology, University Hospital Wales, Cardiff CF14 4XW, UK.

In 2015, the newborn screening (NBS) programmes in England and Wales were expanded to include four additional disorders: Classical Homocystinuria, Isovaleric Acidemia, Glutaric Aciduria Type 1 and Maple Syrup Urine Disease, bringing the total number of analytes quantified to eight: phenylalanine, tyrosine, leucine, methionine, isovalerylcarnitine, glutarylcarnitine, octanoylcarnitine and decanoylcarnitine. Post-implementation, population data monitoring showed that inter-laboratory variation was greater than expected, with 90th centiles varying from 17 to 59%. We evaluated the effect of stable isotope internal standard (IS) used for quantitation on inter-laboratory variation. Four laboratories analysed routine screening samples ( > 101,820) using a common IS. Inter-laboratory variation was determined for the eight analytes and compared with results obtained using an in-house common IS ( > 102,194). A linear mixed-effects model was fitted to the data. Using a common IS mix reduced the inter-laboratory variation significantly ( < 0.05) for five analytes. For three analytes, the lack of significance was explained by use of individual laboratory "calibration factors". For screening programmes where laboratories adhere to single analyte cut-off values (COVs), it is important that inter-laboratory variation is minimised, primarily to prevent false positive results. Whilst the use of a common IS helps achieve this, it is evident that instrument set-up also contributes to inter-laboratory variation.
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http://dx.doi.org/10.3390/ijns6040092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712741PMC
November 2020

Mass Spectrometry Characterization of Higher Order Structural Changes Associated with the Fc-glycan Structure of the NISTmAb Reference Material, RM 8761.

J Am Soc Mass Spectrom 2020 Mar 21;31(3):553-564. Epub 2020 Jan 21.

LGC, Queens Road, Teddington TW11 0LY, UK.

As monoclonal antibodies (mAbs) rapidly emerge as a dominant class of therapeutics, so does the need for suitable analytical technologies to monitor for changes in protein higher order structure (HOS) of these biomolecules. Reference materials (RM) serve a key analytical purpose of benchmarking the suitability and robustness of both established and emerging analytical procedures for both drug producers and regulators. Here, two simple enzymatic protocols for generating Fc-glycan variants from the NISTmAb RM are described and both global and localized changes in HOS between the RM and these Fc-glycan variants are characterized using hydrogen deuterium exchange-mass spectrometry (HDX-MS) and ion mobility spectrometry-mass spectrometry (IMS-MS) measurements. An alternative statistical approach is described where measurement thresholds that differentiate between measurement variability and significant structural changes were established on the basis of experimental data. Measurements revealed decreases in structural stability correlating with the degree of Fc-glycan structure loss, especially at the C2/C3 domain interface. These data promote the use of this RM and these Fc-glycan variants for establishing the sensitivity of and validating analytical methods for the detection of HOS measurements of mAbs.
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http://dx.doi.org/10.1021/jasms.9b00022DOI Listing
March 2020

Lessons learned from inter-laboratory studies of carbon isotope analysis of honey.

Sci Justice 2019 01 16;59(1):9-19. Epub 2018 Aug 16.

Queensland Health Forensic and Scientific Services, P.O. Box 594, Archerfield, Queensland 4108, Australia.

Forensic application of carbon isotope ratio measurements of honey and honey protein to investigate the degree of adulteration with high fructose corn syrup or other C plant sugars is well established. These measurements must use methods that exhibit suitable performance criteria, particularly with regard to measurement uncertainty and traceability - low levels of adulteration can only be detected by methods that result in suitably small measurement uncertainties such that differences of 1‰ or less can be reliably detected. Inter-laboratory exercises are invaluable to assess the state-of-the art of measurement capabilities of laboratories necessary to achieve such performance criteria. National and designated metrology institutes from a number of countries recently participated in an inter-laboratory assessment (CCQM-K140) of stable carbon isotope ratio determination of bulk honey. The same sample material was distributed to a number of forensic isotope analysis laboratories that could not participate directly in the metrological comparison. The results from these studies have demonstrated that the majority of participants provided isotope delta values with acceptable performance metrics; that all participants ensured traceability of their results; and that where measurement uncertainties were reported; these were fit-for-purpose. A number of the forensic laboratories only reported precision rather than full estimates of measurement uncertainty and this was the major cause of the few instances of questionable performance metrics. Reporting of standard deviations in place of measurement uncertainties is common practice outside metrology institutes and the implications for interpretations of small differences in isotopic compositions are discussed. The results have also highlighted a number of considerations that are useful for organisers of similar inter-laboratory studies in the future.
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http://dx.doi.org/10.1016/j.scijus.2018.08.003DOI Listing
January 2019

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

Clin Chem 2018 09 14;64(9):1296-1307. Epub 2018 Jun 14.

Molecular and Cell Biology Team, LGC, Teddington, Middlesex, UK;

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.

Methods: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer ( c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement.

Results: Concentration values for samples of G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively).

Conclusions: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.
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http://dx.doi.org/10.1373/clinchem.2017.285478DOI Listing
September 2018

Feedback from the European Bioanalysis Forum: focus workshop on current analysis of immunogenicity: best practices and regulatory hurdles.

Bioanalysis 2018 Feb 18;10(4):197-204. Epub 2018 Jan 18.

European Bioanalysis Forum, Havenlaan 86c b204, 1000 Brussels, Belgium.

European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.
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http://dx.doi.org/10.4155/bio-2017-4971DOI Listing
February 2018

A generic standard additions based method to determine endogenous analyte concentrations by immunoassays to overcome complex biological matrix interference.

Sci Rep 2017 12 13;7(1):17542. Epub 2017 Dec 13.

LGC, Queens Road, Teddington, UK.

We describe a novel generic method to derive the unknown endogenous concentrations of analyte within complex biological matrices (e.g. serum or plasma) based upon the relationship between the immunoassay signal response of a biological test sample spiked with known analyte concentrations and the log transformed estimated total concentration. If the estimated total analyte concentration is correct, a portion of the sigmoid on a log-log plot is very close to linear, allowing the unknown endogenous concentration to be estimated using a numerical method. This approach obviates conventional relative quantification using an internal standard curve and need for calibrant diluent, and takes into account the individual matrix interference on the immunoassay by spiking the test sample itself. This technique is based on standard additions for chemical analytes. Unknown endogenous analyte concentrations within even 2-fold diluted human plasma may be determined reliably using as few as four reaction wells.
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http://dx.doi.org/10.1038/s41598-017-17823-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727527PMC
December 2017

Almond or Mahaleb? Orthogonal Allergen Analysis During a Live Incident Investigation by ELISA, Molecular Biology, and Protein Mass Spectrometry.

J AOAC Int 2018 Jan 5;101(1):162-169. Epub 2017 Dec 5.

Laboratory of the Government Chemist, Queens Rd, Teddington TW11 0LY, United Kingdom.

It is now well known that an incident investigated in the United Kingdom in 2015 of cumin alleged to be contaminated with almond, a risk for people with almond allergy, was caused by the Prunus species, Prunus mahaleb. In the United Kingdom, the Government Chemist offers a route of technical appeal from official findings in the food control system. Findings of almond in two official samples, cumin and paprika, which had prompted action to exclude the consignments from the food chain, were so referred. Herein are described the approaches deployed to resolve the analytical issues during the investigation of the incidents. The cross-reactivity of ELISA to Prunus species was confirmed, and although this is useful in screening for the genus, orthogonal techniques are required to identify the species and confirm its presence. Two novel PCR assays were developed: one specific for P. mahaleb and the other a screening method capable of identifying common Prunus DNA. Peptides unique to almond and mahaleb were identified, permitting LC-tandem MS and criteria were developed for peptide identification to forensic standards. This work enables a staged approach to be taken to any future incident thought to involve Prunus species and provides a template for the investigation of similar incidents.
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http://dx.doi.org/10.5740/jaoacint.17-0405DOI Listing
January 2018

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia.

Clin Chem 2017 Feb 15;63(2):525-531. Epub 2016 Dec 15.

Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK.

Background: Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1 (%BCR-ABL1) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification.

Methods: BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay.

Results: Measurements on all instruments correlated well when the %BCR-ABL1 was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range.

Conclusions: RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.
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http://dx.doi.org/10.1373/clinchem.2016.262824DOI Listing
February 2017

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

Anal Chem 2017 02 18;89(3):1724-1733. Epub 2017 Jan 18.

Dana Farber Cancer Institute , Belfer Center for Applied Cancer Science and Department of Medical Oncology, Boston, Massachusetts 02115, United States.

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. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.
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http://dx.doi.org/10.1021/acs.analchem.6b03980DOI Listing
February 2017

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

Biomol Detect Quantif 2016 Jun 6;8:15-28. Epub 2016 Jun 6.

National Institute of Metrology (NIM), Beijing, PR China.

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. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.
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http://dx.doi.org/10.1016/j.bdq.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906133PMC
June 2016

Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

J Clin Microbiol 2016 Feb 9;54(2):392-400. Epub 2015 Dec 9.

Molecular and Cell Biology Team, LGC, Teddington, United Kingdom Department of Infection, Division of Infection and Immunity, University College London, London, United Kingdom.

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.
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http://dx.doi.org/10.1128/JCM.02611-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733194PMC
February 2016

Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS.

Clin Chem 2016 Jan 3;62(1):262-9. Epub 2015 Nov 3.

University Hospital South Manchester, Manchester, UK; Manchester Academic Health Science Centre, Manchester, UK;

Background: Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results.

Method: Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result.

Results: Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ionization efficiency studies and structural analog analysis proved the method to be robust against interferences. Through analysis of 34 CRMs (83-764 nmol/L), expanded measurement uncertainty was calculated to be 5% (95% CI). The mean bias between the measured and target CRM concentrations was statistically insignificant at -0.08%.

Conclusions: The accuracy and low measurement uncertainty of this method qualify it as a CRM procedure. Metrological traceability has been achieved through the analysis of higher-order CRMs. This method could be used to underpin serum cortisol EQA schemes to provide samples with a traceable target value, enabling participating laboratories to determine the accuracy and measurement uncertainty of their assays.
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http://dx.doi.org/10.1373/clinchem.2015.243576DOI Listing
January 2016

Considerations for digital PCR as an accurate molecular diagnostic tool.

Clin Chem 2015 Jan 22;61(1):79-88. Epub 2014 Oct 22.

LGC, Teddington, UK;

Background: Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described.

Content: This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence.

Summary: In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.
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http://dx.doi.org/10.1373/clinchem.2014.221366DOI Listing
January 2015

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

Anal Bioanal Chem 2014 Oct 24;406(26):6499-512. Epub 2014 May 24.

Molecular and Cell Biology Team, LGC Ltd, Queen's Road, Teddington, TW11 0LY, UK,

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.
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http://dx.doi.org/10.1007/s00216-014-7835-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182654PMC
October 2014

A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability.

Biotechniques 2013 Mar;54(3):155-64

LGC Limited, Queens Road, Teddington, Middlesex, UK.

MicroRNAs (miRNAs) are short (~22 nucleotides), non-coding RNA molecules that post-transcriptionally regulate gene expression. As the miRNA field is still in its relative infancy, there is currently a lack of consensus regarding optimal methodologies for miRNA quantification, data analysis and data standardization. To investigate miRNA measurement we selected a panel of both synthetic miRNA spikes and endogenous miRNAs to evaluate assay performance, copy number estimation, and relative quantification. We compared two different miRNA quantification methodologies and also assessed the impact of short RNA enrichment on the miRNA measurement. We found that both short RNA enrichment and quantification strategy used had a significant impact on miRNA measurement. Our findings illustrate that miRNA quantification can be influenced by the choice of methodology and this must be considered when interpreting miRNA analyses. Furthermore, we show that synthetic miRNA spikes can be used as effective experimental controls for the short RNA enrichment procedure.
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http://dx.doi.org/10.2144/000114002DOI Listing
March 2013

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

PLoS One 2013 5;8(3):e58177. Epub 2013 Mar 5.

Molecular and Cell Biology Team, LGC Ltd, Teddington, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058177PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589384PMC
December 2013

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

Nucleic Acids Res 2012 Jun 28;40(11):e82. Epub 2012 Feb 28.

LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.

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.
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http://dx.doi.org/10.1093/nar/gks203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367212PMC
June 2012

Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry.

Forensic Sci Int Genet 2013 Jan 17;7(1):1-9. Epub 2011 Jun 17.

LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.

Mitochondrial DNA is commonly used in identity testing for the analysis of old or degraded samples or to give evidence of familial links. The Abbott T5000 mass spectrometry platform provides an alternative to the more commonly used Sanger sequencing for the analysis of human mitochondrial DNA. The robustness of the T5000 system has previously been demonstrated using DNA extracted from volunteer buccal swabs but the system has not been tested using more challenging sample types. For mass spectrometry to be considered as a valid alternative to Sanger sequencing it must also be demonstrated to be suitable for use with more limiting sample types such as old teeth, bone fragments, and hair shafts. In 2009 the Commonwealth War Graves Commission launched a project to identify the remains of 250 World War I soldiers discovered in a mass grave in Fromelles, France. This study characterises the performance of both Sanger sequencing and the T5000 platform for the analysis of the mitochondrial DNA extracted from 225 of these remains, both in terms of the ability to amplify and characterise DNA regions of interest and the relative information content and ease-of-use associated with each method.
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http://dx.doi.org/10.1016/j.fsigen.2011.05.009DOI Listing
January 2013

Evaluation of digital PCR for absolute DNA quantification.

Anal Chem 2011 Sep 1;83(17):6474-84. Epub 2011 Aug 1.

LGC, Queens Road, Teddington, Middlesex TW11 0LY.

The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.
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http://dx.doi.org/10.1021/ac103230cDOI Listing
September 2011

An investigation of the robustness of the consensus method of interpreting low-template DNA profiles.

Forensic Sci Int Genet 2011 Nov 16;5(5):400-6. Epub 2010 Oct 16.

LGC Forensics, Queens Road, Teddington, Middlesex TW11 0LY, UK.

Forensic STR profiles generated from low-template DNA samples are more noticeably subject to effects such as allele dropout, contamination with spurious alleles ('drop-in') and proportionally larger stutter. The profiles obtained are frequently partial, and are challenging to interpret. Specifically, interpretation guidelines which are used when the template DNA is in the optimal range for the STR test kit in use must be adapted to the low-template regime. A commonly used approach to such modified interpretation is known as the 'consensus' or 'biological' method, and relies on replication to achieve reliable results. We have carried out a study to assess the robustness of the consensus method as applied to SGM Plus(®) STR profiles obtained after applying a set of post-PCR purification methods together known as DNA SenCE, and report the results here. Multiple repeat analysis of DNA at five template quantities (ranging between 5pg and 100pg) and from five single donors, was carried out, and the resulting profiles were used to produce consensus profiles using several different evaluation criteria. Our aim was to determine whether the consensus profiles produced are conservative, that is, that the alleles reported are associated with the donor and that drop-in is reduced or eliminated. To this end, the alleles in the consensus profiles were compared with those of the donors, and the degree of concordance determined. The results suggest that increasingly stringent requirements for the number of times an allele must be observed in a set of repeat runs do, as expected, reduce the effect of drop-in, but also reduce the evidential value of the consensus profiles. However, the former is reduced to a much greater extent than the latter, resulting in a relative increase in profile information content versus drop-in peak risk with increased stringency. We also found that approximately half of the non-donor peaks appearing in consensus profiles were in -4 stutter positions for donor alleles present in the same profile, suggesting that many of these so-called drop-in alleles are, in fact, large stutter peaks rather than 'true' drop-in. Nevertheless, the appearance of non-donor peaks in a profile, including what are assumed to be oversized stutter peaks, appears to be an essentially random event.
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http://dx.doi.org/10.1016/j.fsigen.2010.08.010DOI Listing
November 2011

Reporting measurement uncertainty and coverage intervals near natural limits.

Analyst 2006 Jun 11;131(6):710-7. Epub 2006 May 11.

LGC Limited, Queens Road, Teddington, Middlesex, UK TW11 0LY.

Different methods of treating data which lie close to a natural limit in a feasible range, such as zero or 100% mass or mole fraction, are discussed and recommendations made concerning the most appropriate. The methods considered include discarding observations beyond the limit, shifting observations to the limit, truncation of a classical confidence interval based on Student's t (coupled with shifting the result to the limit if outside the feasible range), truncation and renormalisation of an assumed normal distribution, and the maximum density interval of a Bayesian estimate based on a normal measurement distribution and a uniform prior within the feasible range. Based on consideration of bias and simulation to assess coverage, it is recommended that for most purposes, a confidence interval near a natural limit should be constructed by first calculating the usual confidence interval based on Student's t, then truncating the out-of-range portion to leave an asymmetric interval and adjusting the reported value to within the resulting interval if required. It is suggested that the original standard uncertainty is retained for uncertainty propagation purposes.
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http://dx.doi.org/10.1039/b518084hDOI Listing
June 2006