Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, ‘definetherain’

Highlights • The requirement for protocol adaptation from qPCR to ddPCR is characterised.• A potential loss of sensitivity for ddPCR at low target numbers is reported.• A new bioinformatic tool ‘definetherain’ to improve droplet calling at low input target numbers is devised and applied.• ‘Definetherain’ is made available free of charge and open access at www.definetherain.org.uk.

[1]  Linos Vandekerckhove,et al.  Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA , 2014, PloS one.

[2]  Benjamin J. Hindson,et al.  Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification , 2011, Analytical chemistry.

[3]  Z. Gu,et al.  Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus , 2012, Journal of Clinical Microbiology.

[4]  Douglas D. Richman,et al.  Highly Precise Measurement of HIV DNA by Droplet Digital PCR , 2013, PloS one.

[5]  Sebastien Gallien,et al.  Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. , 2012, Journal of virological methods.

[6]  R Brookmeyer,et al.  Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. , 1997, Science.

[7]  D. Richman,et al.  Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. , 1997, Science.

[8]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[9]  R. Siliciano,et al.  Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies , 2013, PLoS pathogens.

[10]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[11]  Hanlee P. Ji,et al.  Correction to High Sensitivity Detection and Quantitation of DNA Copy Number and Single Nucleotide Variants with Single Color Droplet Digital PCR , 2015, Analytical chemistry.