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Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR Alison S. Devonshire, Isobella Honeyborne, Alice Gutteridge, Alexandra S. Whale, Gavin Nixon, Philip Wilson, Gerwyn Jones, Timothy D. Mchugh, Carole A. Foy, and Jim Francis Huggett Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 03 Feb 2015 Downloaded from http://pubs.acs.org on February 4, 2015
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Analytical Chemistry
Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR. Alison S. Devonshire1, Isobella Honeyborne2, Alice Gutteridge1†, Alexandra S. Whale1, Gavin Nixon1, Philip Wilson3, Gerwyn Jones1, Timothy D. McHugh2, Carole A. Foy1 and Jim F. Huggett1&2*. 1
Molecular and Cell Biology team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom
2
Centre for Clinical Microbiology, Department of Infection, Royal Free Campus, University College London, London NW3 2PF, United Kingdom
3
Statistics team, LGC, Teddington, Middlesex TW11 0LY, United Kingdom.
ABSTRACT: 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, mastermixes, primer pairs and, crucially, extraction methods on dPCR performance. Performance was compared between the chip (BioMark) and droplet (QX100) formats. In the absence of any external calibration dPCR measurements were generally consistent within ~2-fold between different mastermixes and primers. Template DNA integrity could influence dPCR performance: high molecular weight gDNA resulted in under-performance of one mastermix, while restriction digestion of a low molecular weight sample also caused underestimation. Good concordance (≤ 1.5-fold difference) was observed between chip and droplet formats. Platform precision was in agreement with predicted Poisson error based on partition number but this was a minor component ( 4.0 kbp) and plasmid (8.0 kbp) templates (Figure S2). To test this hypothesis, restriction digestion of H37Ra and H37Rv gDNA with NarI was performed which resulted in fragmentation of both templates to < 4.0 kbp (Figure S3). The dPCR performance of the Biotium mastermix with non-digested and NarI-digested templates was compared with GEMM. Restriction digestion of H37Ra gDNA increased the apparent DNA copy number measured using the Biotium mastermix whereas this did not influence dPCR measurements using GEMM (Figure 1D). Restriction digestion of H37Rv gDNA resulted in a decrease in the number of copies measured by both Biotium and GEMM with no significant difference between mastermixes (Figure 1E). Further analysis of dPCR amplification (Cq data) was performed to investigate whether DNA conformation may influence copy number estimation. Measured values of H37Rv gDNA were significantly higher than those of plasmid and H37Ra gDNA (Figure 1A-C) despite samples being of approximately equal concentration as determined using the Qubit® Fluorometer. Modelling of the frequency distribution of Cq values 25 revealed that H37Rv, unlike plasmid and H37Ra gDNA, displayed an amplification pattern which was not consistent with the observed λ values and doubling with every PCR cycle (Figure S8). The Cq distribution for H37Rv may be characteristic of some of the molecules being present in single stranded conformation, which would be predicted to undergo doubling one cycle later than double-stranded DNA, resulting in an additional peak in the Cq distribution. The impact of dPCR primers location on copy number enumeration of the three template types was compared, for GEMM (Figure 2) and the Kapa, Biotium and BioRad mastermixes (Figure S9). For plasmid and H37Ra DNA, measured values with GN_rpoB2 were consistently lower than the other primers tested (significant at the 95% confidence level) (Figure 2A-B). For H37Rv gDNA, both sets of primers to 16S rRNA produced a higher copy number estimate than those to rpoB (95% level) (Figure 2C). It was verified that the reduced copy number values associated with GN_rpoB2 primers were not due to base mismatches in primer or probe by sequencing of H37Ra and H37Rv gDNA (Supplementary Data). In summary the largest difference measured for an identical template was approximately two fold which
primarily reflected reduced performance of the Biotium mastermix; there was also a smaller ~1.2 fold decrease for primer combination GN_rpoB2 in comparison to all other primer pairs. A previous comparison of mastermixes from a single manufacturer also found highly consistent results (
