Applications of Digital PCR

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Overview

Digital PCR is a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. This technique is particularly useful for low abundance targets, targets in complex backgrounds, allelic variants (SNPs) and for monitoring of subtle changes in target levels.

In the Droplet Digital™ PCR (ddPCR™) System, each PCR sample is partitioned into a large number of microscopic droplets prior to amplification. Each droplet is an individual PCR reaction. After end-point amplification, fluorescence is detected in the droplets in which target sequence was amplified. These droplets are scored as positive. Droplets not containing the target sequence show little or no fluorescence and are scored as negative. Using the Poisson distribution law, the fraction of positive droplets is converted to the number of molecules in the starting sample, without the need for standard curves (absolute quantification).

Applications for digital PCR cover different areas of biology. The following describes some of the most popular fields of application.

Liquid Biopsy

Liquid biopsies are noninvasive tests that are able to detect cancer cells (circulating tumor cells, CTCs) or DNA shed from tumors (circulating tumor DNA, ctDNA) in the blood. They are increasingly used for cancer detection and monitoring. Unlike tumor tissue biopsies, liquid biopsies present minimal risk to a person and they can be readily repeated over time for the purposes of diagnosing and quantifying disease, and monitoring its progress.

Target ctDNA and CTCs are present at low levels in a complex background of cell-free DNA (cfDNA). Given its high level of sensitivity, Droplet Digital PCR is able to accurately detect and quantify rare sequences in the presence of abundant targets. A range of studies have shown that ddPCR can be used for liquid biopsy, often with higher sensitivity than other techniques.

This technique is not only useful for the detection of different cancers, but it can also be used to monitor changes in a cancer, detect tumor heterogeneity, find biomarkers, and detect loss of response to treatment. The range of diseases that can be detected and tracked by liquid biopsy is rapidly expanding. Some examples include early detection of type 1 diabetes, detection and monitoring of infections, organ transplantation, and noninvasive pregnancy testing.

Learn more about Droplet Digital PCR and Liquid Biopsy.

Copy Number Variation (CNV)

Estimates suggest that over 10% of the human genome is composed of CNVs with sequences larger than 1 kb, and that about 30% of the reference human genome contains CNVs of sequences larger than 100 bp. Some regions can have many copies per cell. A number of CNVs have been linked to diseases. Disease-associated CNVs can be inherited or generated de novo. In addition to diseases, CNVs in dosage-sensitive genes are associated with complex phenotypic and behavior traits. The ddPCR System has been demonstrated to be well suited to high-throughput studies profiling CNV variations in populations of interest (Karlin-Neumann et al. 2011, Boettger et al. 2012).

Digital PCR can provide accurate CNV quantification in single wells. In qPCR assays, many replicates are needed to accurately discriminate CNVs. For example, to ascertain a copy number change from 4 to 5, up to 18 qPCR replicates are required. The precision and sensitivity of ddPCR technology allows the system to distinguish small changes much more readily: the same change from four to five copies can be determined using just one well. Additionally, the accuracy of digital PCR is less sensitive to changes in amplification efficiency, a major cause of inaccuracy in qPCR measurements.

Learn more about Digital PCR for Copy Number Variation Analysis.

Rare Sequence Detection

Increasing detection sensitivity for low-abundance targets will have broad impacts ranging from earlier and more precise detection of cellular changes in research and clinical settings to the development of a wider array of noninvasive tests.

Digital PCR allows for increased performance in the detection and quantitation of rare sequences because target quantification is independent of the number of amplification cycles. Moreover, when detecting related sequences (SNPs, allelic variants, edited RNA), partitioning reduces competition with the more abundant background (wild-type) species. Expanding uses of digital PCR for rare sequence detection include:

  • Detection of cancer below the level detectable by current tests
  • Monitoring for new mutations and duplications in cancer as they arise
  • Detection of viral loads, such as HIV, below those detectable by current testing
  • Noninvasive testing in bodily fluids for infectious diseases and cancer, including circulating cell-free DNA (cfDNA)
  • Noninvasive prenatal testing using cfDNA
  • Detection of transplant rejection in circulating DNA

Learn more about Rare Sequence Detection Using Digital PCR.

Gene Expression

The increased precision of digital PCR can provide higher resolution in many aspects of gene expression measurement. It enables scientists to precisely quantify finer changes in expression levels (less than twofold). Digital PCR also provides a greater sensitivity when quantifying rare targets, or RNA from very limited material.

Digital PCR is used for the analysis of gene expression at both the DNA and RNA levels in the following areas:

  • Detection and measurement of genomic DNA methylation
  • Increased sensitivity in transcriptional analyses with absolute quantification
  • Detection of rare mRNAs and miRNAs with rapid turnover including those in complex matrices such as blood
  • Detection of as few as 2 targets per well using EvaGreen with the QX200™ Droplet Digital PCR System. This allows users to perform gene expression analysis without TaqMan probes.

Learn more about Gene Expression and Digital PCR.

Single-Cell Analysis

Single-cell PCR is challenging. Not only can the isolation of an intact single cell be difficult, but getting accurate results from the low levels of starting material has been technically challenging.

The following characteristics of digital PCR make it a sensitive and robust tool for single-cell analysis:

  • Precise detection and amplification of targets at low template levels
  • No requirement for a standard curve or housekeeping genes
  • Reduced sensitivity to PCR-inhibiting components in crude cell lysates
  • Simultaneous detection of 4 targets through multiplexing
  • Can be combined with other techniques such as next-generation sequencing

Learn more about Single-Cell Analysis Using Digital PCR.

Pathogen Detection and Microbiome Analysis

Perhaps the most extensive use of digital PCR to date has been in microbiology. Both pathogen detection and microbiome analysis often require the detection and quantitation of low-abundance microorganisms in complex backgrounds.

Some areas of pathogen detection in which digital PCR is used are:

  • Detection and monitoring of viral loads that are below the limit of quantification of current tests
  • Pathogen detection both pre- and post-allotransplantation
  • Detection and monitoring of microbial drug resistance and heteroresistance
  • Detection of pathogens in food

The analysis of microbiomes can be difficult and labor intensive due to the large number of species and subspecies in most biomes and their often continually changing nature. The high sensitivity, ability to amplify rare targets in complex backgrounds, and reduced sensitivity to PCR inhibitors such as humic acid are all features of digital PCR that can improve microbiome analysis. Uses of digital PCR include:

  • Tracking changes in population composition
  • Cosegregation analysis including phylogenetic relationships

Learn more about Pathogen Detection and Microbiome Analysis Using Digital PCR.

Next-Generation Sequencing (NGS)

Digital PCR can increase the efficiency and accuracy of NGS, saving both money and time.

Integration of digital PCR into an NGS workflow can occur at several steps:

  • Amplification of target libraries, ensuring better representation of low abundance species
  • Accurate quantification of NGS libraries
  • Validation of sequencing results

Learn more about Digital PCR and Next-Generation Sequencing.

References

Boettger LM et al. (2012). Structural haplotypes and recent evolution of the human 17q21.31 region. Nat Genet 44, 881–885. PMID: 22751096

Karlin-Neumann G et al. (2011). Probing copy number variations using Bio-Rad's QX100™
Droplet Digital™ PCR System. Bio-Rad Bulletin 6277.

For Further Reading — Droplet Digital™ PCR Publications

Browse the growing list of research articles featuring Bio-Rad's Droplet Digital PCR technology:

Abdel-Wahab O et al. (2014). Efficacy of intermittent combined RAF and MEK inhibition in a patient with concurrent BRAF- and NRAS-mutant malignancies. Cancer Discov 4, 538–545. PMID: 24589925

Abyzov A et al. (2012). Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature 492, 438–442. PMID: 23160490

Agapova S et al. (2013). Detection of low-concentration host mRNA transcripts in Malawian children at risk for environmental enteropathy. J Pediatr Gastroenterol Nutr 56, 66–71. PMID: 22832511

Antas VI et al. (2014). Gastrokine-2 is transiently expressed in the endodermal and endothelial cells of the maturing mouse yolk sac. Gene Expr Patterns 16, 69–74. PMID: 25290738

Arendt M et al. (2014). Amylase activity is associated with AMY2B copy numbers in dog: Implications for dog domestication, diet, and diabetes. Anim Genet 45, 716–722. PMID: 24975239

Aubert M et al. (2014). In vitro inactivation of latent HSV by targeted mutagenesis using an HSV- specific homing endonuclease. Mol Ther Nucleic Acids 3, e146. PMID: 24496438

Bahn JH et al. (2015). The landscape of microRNA, piwi-interacting RNA, and circular RNA in human saliva. Clin Chem 61, 221–230. PMID: 25376581

Baker M (2012). Digital PCR hits its stride. Nat Methods 9, 541–544.

Baoutina A et al. (2013). Improved detection of transgene and nonviral vectors in blood. Hum Gene Ther Methods 24, 345–354. PMID: 23971678

Beaver JA et al. (2014). Detection of cancer DNA in plasma of patients with early-stage breast cancer. Clin Cancer Res 20, 2,643–2,650. PMID: 24504125

Beck J et al. (2013). Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA. PLoS One 8, e75485. PMID: 24098698

Belgrader P et al. (2013). Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue. Clin Chem 59, 991–994. PMID: 23358413

Beliakova-Bethell N et al. (2014). Maraviroc intensification in patients with suppressed HIV viremia has limited effects on CD4+ T cell recovery and gene expression. Antiviral Res 107, 42–49. PMID: 24769244

Beliakova-Bethell N et al. (2014). The effect of cell subset isolation method on gene expression in leukocytes. Cytometry A 85, 94–104. PMID: 24115734

Belzil VV et al. (2013). Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood. Acta Neuropathol 126, 895–905. PMID: 24166615

Bharuthram A et al. (2014). Comparison of a quantitative real-time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes. Infect Genet Evol 25, 28–35. PMID: 24727646

Bizouarn F (2012). Digital PCR: Improving nucleic acid quantification — precision, accuracy, and sensitivity are among the benefits reported by researchers. Genetic Engineering & Biotechnology News 32, 9.

Bizouarn F (2014). Clinical applications using digital PCR. Methods Mol Biol. 1,160:189–214. PMID: 24740231

Bizouarn F (2014). Introduction to digital PCR. Methods Mol Biol. 1,160:27–41. PMID: 24740219

Blevins T et al. (2014). A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54, 30–42. PMID: 24657166

Boettger LM et al. (2012). Structural haplotypes and recent evolution of the human 17q 21.31 region. Nat Genet 44, 881– 885. PMID: 22751096

Boizeau L et al. (2014). Could droplet digital PCR be used instead of real-time PCR for quantitative detection of the hepatitis B virus genome in plasma? J Clin Microbiol 52, 3,497–3,498. PMID: 24989610

Bozdag GO and Greig D (2014). The genetics of a putative social trait in natural populations of yeast. Mol Ecol 23, 5,061–5,071. PMID: 25169714

Brunetto GS et al. (2014). Digital droplet PCR (ddPCR) for the precise quantification of human T- lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations. J Neurovirol 20, 341–351. PMID: 24781526

Cai J et al. (2014). Whole-genome sequencing identifies genetic variances in culture-expanded human mesenchymal stem cells. Stem Cell Reports 3, 227–233. PMID: 25254336

Cai Y et al. (2014). Quantitative analysis of pork and chicken products by droplet digital PCR. BioMed Res Int 2014, 810209. PMID: 25243184

Campbell IM et al. (2014). Parental somatic mosaicism is under-recognized and influences recurrence risk of genomic disorders. Am J Hum Genet 95, 173–182. PMID: 25087610

Cangi MG et al. (2014). BRAFV600E-mutation is invariably present and associated to oncogene- induced senescence in Erdheim-Chester disease. Ann Rheum Dis, Mar 26 [Epub ahead of print]. Accessed January 29, 2015. PMID: 24671772

Carreira S et al. (2014). Tumor clone dynamics in lethal prostate cancer. Sci Transl Med 6, 254ra125. PMID: 25232177

Chen R et al. (2012). Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell 148, 1,293–1,307. PMID: 22424236

Choi YP et al. (2014). Cancer-associated fibroblasts promote transmigration through endothelial brain cells in three-dimensional in vitro models. Int J Cancer 135, 2,024–2,033. PMID: 24643985

Chong IY et al. (2013). The genomic landscape of oesophagogastric junctional adenocarcinoma. J Pathol 231, 301–310. PMID: 24308032

Cochran RL et al. (2014). Analysis of BRCA2 loss of heterozygosity in tumor tissue using droplet digital polymerase chain reaction. Hum Pathol 45, 1,546–1,550. PMID: 24824029

Criscione F et al. (2013). A unique Y gene in the Asian malaria mosquito Anopheles stephensi encodes a small lysine-rich protein and is transcribed at the onset of embryonic development. Insect Mol Biol 22, 433–441. PMID: 23683123

Davis JM et al. (2014). DUF1220 dosage is linearly associated with increasing severity of the three primary symptoms of autism. PLoS Genet 10, e1004241. PMID: 24651471

Devonshire AS et al. (2014). Towards standardisation of cell-free DNA measurement in plasma: Controls for extraction efficiency, fragment size bias, and quantification. Anal Bioanal Chem 406, 6,499–6,512. PMID: 24853859

Dingle TC et al. (2013). Tolerance of droplet-digital PCR vs. real-time quantitative PCR to inhibitory substances. Clin Chem 59, 1,670–1,672. PMID: 24003063

Dodd DW et al. (2013). Digital quantitation of potential therapeutic target RNAs. Nucleic Acid Ther 23, 188–194. PMID: 23656494

Dong L et al. (2014). Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers. Anal Bioanal Chem 406, 1,701–1,712. PMID: 24493332

Dreo T et al. (2014). Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: A case study of fire blight and potato brown rot. Anal Bioanal Chem 406, 6,513–6,528. PMID: 25173868

Emerson RO et al. (2013). High-throughput sequencing of T cell receptors reveals a homogeneous repertoire of tumor-infiltrating lymphocytes in ovarian cancer. J Pathol 231, 433–440. PMID: 24027095

Eriksson S et al. (2013). Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog 9, e1003174. PMID: 23459007

Floren C et al. (2015). Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR). Food Chem 173, 1,054–1,058. PMID: 25466124

Frésard L et al. (2014). Transcriptome-wide investigation of genomic imprinting in chicken. Nucleic Acids Res 42, 3,768–3,782. 24452801

Furlan D et al. (2014). APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation. Virchows Arch 464, 553–564. PMID: 24590585

Gagnon P et al. (2014). Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance. J Chromatogr A 1340, 68–78. PMID: 24661871

Gangoda L et al. (2014). Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice. Cell Death Differ 21, 1,815–1,824. PMID: 25012505

Garcia-Murillas I et al. (2013). Determination of HER2 amplification status on tumour DNA by digital PCR. PLoS One 8, e83409. PMID: 24386193

Gevensleben H et al. (2013). Noninvasive detection of HER2 amplification with plasma DNA digital PCR. Clin Cancer Res 19, 3,276–3,284. 23637122

Gianella S et al. (2014). Cytomegalovirus replication in semen is associated with higher levels of proviral HIV DNA and CD4+ T cell activation during antiretroviral treatment. J Virol 88, 7,818–7,827. PMID: 24789781

Glessner JT et al. (2014). Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data. Circ Res 115, 884–896. PMID: 25205790

Gorbachev AY et al. (2013). DNA repair in Mycoplasma gallisepticum. BMC Genomics 14, 726. PMID: 24148612

Gu W et al. (2014). Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia. Genet Med 16, 564–567. PMID: 24406457

Hall Sedlak R and Jerome KR (2014). The potential advantages of digital PCR for clinical virology diagnostics. Expert Rev Mol Diagn 14, 501–507. PMID: 24724628

Hashimoto-Torii K et al. (2014). Roles of heat shock factor 1 in neuronal response to fetal environmental risks and its relevance to brain disorders. Neuron 82, 560–572. PMID: 24726381

Hatano H et al. (2013). Increase in 2-long terminal repeat circles and decrease in D-dimer after raltegravir intensification in patients with treated HIV infection: A randomized, placebo-controlled trial. J Infect Dis 208, 1,436–1,442. PMID: 23975885

Hayden RT et al. (2013). Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. J Clin Microbiol 51, 540–546. PMID: 23224089

Henrich TJ et al. (2012). Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. J Virol Methods 186, 68–72. PMID: 22974526

Heredia NJ et al. (2013). Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods 59, S20–S23. PMID: 23036330

Hill JA et al. (2014). Hepatitis due to human herpesvirus 6B after hematopoietic cell transplantation and a review of the literature. Transpl Infect Dis 16, 477–483. PMID: 24703390

Hindson BJ et al. (2011). High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83, 8,604–8,610. PMID: 22035192

Hindson CM et al. (2013). Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10, 1,003–1,005. PMID: 23995387

Holmberg RC et al. (2013). Akonni TruTip® and Qiagen® methods for extraction of fetal circulating DNA — evaluation by real-time and digital PCR. PLoS One 8, e73068. PMID: 23936545

Huang JT et al. (2015). Next generation digital PCR measurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue. Clin Chem 61, 290–296. PMID: 25361948

Hubers AJ et al. (2013). EGFR mutation analysis in sputum of lung cancer patients: A multitechnique study. Lung Cancer 82, 38–43. PMID: 23927883

Huggett JF et al. (2015). Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 61, 79–88. PMID: 25338683

Hwang VJ et al. (2014). Mappng the deletion endpoints in individuals with 22q11.2 deletion syndrome by droplet digital PCR. BMC Med Genet 15, 106. PMID: 25312060

Iyer CC et al. (2014). Deletion of atrophy enhancing genes fails to ameliorate the phenotype in a mouse model of spinal muscular atrophy. Neuromuscul Disord 24, 436–444. PMID: 24656734

Izumi K et al. (2014). 12p microRNA expression in fibroblast cell lines from probands with Pallister-Killian syndrome. Chromosome Res 22, 453–461. PMID: 24981202

Jahn M et al. (2014). Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR. Anal Chem 86, 5,969–5,976. PMID: 24842041

Jennings LJ et al. (2014). Detection and quantification of BCR-ABL1 fusion transcripts by droplet digital PCR. J Mol Diagn 16, 174–179. PMID: 24389534

Jiang K et al. (2013). MicroRNA-137 represses Klf4 and Tbx3 during differentiation of mouse embryonic stem cells. Stem Cell Res 11, 1,299–1,313. PMID: 24084696

Johnson BE et al. (2014). Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 343, 189–193. PMID: 24336570

Jones M et al. (2014). Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, “definetherain.” J Virol Methods 202, 46–53. PMID: 24598230

Kay MA and Walker BD (2014). Engineering cellular resistance to HIV. N Engl J Med 370, 968–969. PMID: 24597871

Kelley K et al. (2013). Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay. J Clin Microbiol 51, 2,033–2,039. PMID: 23596244

Kim TG et al. (2014). Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil. Appl Microbiol Biotechnol 98, 6,105–6,113. PMID: 24831026

Kim TG et al. (2014). Comparison of droplet digital PCR and quantitative real-time PCR in mcrA-based methanogen community analysis. Biotechnology Reports 4, 1–4.

Kim TG et al. (2015). Development of droplet digital PCR assays for methanogenic taxa and examination of methanogen communities in full-scale anaerobic digesters. Appl Microbiol Biotechnol 9 9, 445–458. PMID: 25117548

Kiselinova M et al. (2014). Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA. PLoS One 9, e85999. PMID: 24465831

Koren A et al. (2014). Genetic variation in human DNA replication timing. Cell 159, 1,015–1,026. PMID: 25416942

Last AR et al. (2014). Plasmid copy number and disease severity in naturally occurring ocular Chlamydia trachomatis infection. J Clin Microbiol 52, 324–327. PMID: 24197878

Laurie MT et al. (2013). Simultaneous digital quantification and fluorescence-based size characterization of massively parallel sequencing libraries. BioTechniques 55, 61–67.

Leibovitch EC et al. (2014). Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS One 9, e92328. PMID: 24281335

Li N et al. (2014). Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer. J Cancer Res Clin Oncol 140, 145–150. PMID: 24281335

Lock M et al. (2014). Absolute determination of single-stranded and self-complementary adeno- associated viral vector genome titers by droplet digital PCR. Hum Gene Ther Methods 25, 115–125. PMID: 24328707

López-Knowles E et al. (2014). Relationship of PIK3CA mutation and pathway activity with anti-proliferative response to aromatase inhibition. Breast Cancer Res 16, R68. PMID: 24981670

Ludlow AT et al. (2014). Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution. Nucleic Acids Res 42, e104. PMID: 24861623

Lui YL and Tan EL (2014). Droplet digital PCR as a useful tool for the quantitative detection of enterovirus 71. J Virol Methods 207, 200–203. PMID: 25064359

Lundberg IV et al. (2014). SOX2 expression is regulated by BRAF and contributes to poor patient prognosis in colorectal cancer. PLoS One 9, e101957. PMID: 25010701

Luzuriaga K et al. (2014). HIV type 1 (HIV-1) proviral reservoirs decay continuously under sustained virologic control in HIV-1-infected children who received early treatment. J Infect Dis 210, 1,529–1,538. PMID: 24850788

Ma J et al. (2013). Quantification of plasma miRNAs by digital PCR for cancer diagnosis. Biomark Insights 14, 127–136. PMID: 24277982

Manoj P (2014). Droplet digital PCR technology promises new applications and research areas. Mitochondrial DNA, Apr 29 [Epub ahead of print]. Accessed January 29, 2015. PMID: 24779593

Manokhina I et al. (2014). Quantification of cell-free DNA in normal and complicated pregnancies: Overcoming biological and technical issues. PLoS One 9, e101500. PMID: 24987984

Marques FZ et al. (2014). Measurement of absolute copy number variation reveals association with essential hypertension. BMC Med Genomics 7, 44. PMID: 25027169

Maruyama Y et al. (2014). LC3B is indispensable for selective autophagy of p62 but not basal autophagy. Biochem Biophys Res Commun 446, 309–315. PMID: 24582747

Massanella M et al. (2013). Differential gene expression in HIV-infected individuals following ART. Antiviral Res 100, 420–428. PMID: 23933117

Mazaika E and Homsy J (2014). Digital droplet PCR: CNV analysis and other applications. Curr Protoc Hum Genet 82, 7.24.1–7.24.13. PMID: 25042719

McDermott GP et al. (2013). Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR. Anal Chem 85, 11,619–11,627. PMID: 24180464

McDermott GP et al. (2013). Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR. Anal Chem 85(23):11,619–27. PMID: 24180464

Mehle N et al. (2014). Quantitative analysis of “flavescence doreé” phytoplasma with droplet digital PCR. Phytopathogenic Mollicutes 4, 9–15.

Melchor L et al. (2014). Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models. J Pathol 233, 124–137. PMID: 24615332

Milavec M et al. (2014). GMO quantification: Valuable experience and insights for the future. Anal Bioanal Chem 406, 6,485–6,497. PMID: 25182968

Miotke L et al. (2014). High-sensitivity detection and quantitation of DNA copy number and single-nucleotide variants with single-color droplet digital PCR. Anal Chem 86, 2,618–2,624. PMID: 24483992

Miotto E et al. (2014). Quantification of circulating miRNAs by droplet digital PCR: Comparison of EvaGreen- and TaqMan-based chemistries. Cancer Epidemiol Biomarkers Prev 23, 2,638–2,642. PMID: 25472671

Mitsui T et al. (2014). Comprehensive next-generation sequencing analyses of hypoparathyroidism: Identification of novel GCM2 mutations. J Clin Endocrinol Metab 99, E2,421–E2,428. PMID: 25137426

Morisset D et al. (2013). Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One 8, e62583. PMID: 23658750

Moser DA et al. (2014). Transgene detection by digital droplet PCR. PLoS One 9, e111781. PMID: 25375130

Moser O et al. (2014). Sustained complete molecular remission after imatinib discontinuation in children with chronic myeloid leukemia. Pediatr Blood Cancer 61, 2,080–2,082. PMID: 24810322

Mukaide M et al. (2014). High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70. J Virol Methods 207, 169–177. PMID: 25019167

Nadauld L et al. (2012). Quantitative and sensitive detection of cancer genome amplifications from formalin fixed paraffin embedded tumors with droplet digital PCR. Transl Med 2(2). PMID: 23682346

Nadauld LD et al. (2014). Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer. Genome Biol 15, 428. PMID: 25315765

Nair VD et al. (2012). Involvement of histone demethylase LSD1 in short-time-scale gene expression changes during cell cycle progression in embryonic stem cells. Mol Cell Biol 32, 4,861–4,876. PMID: 23028048

Nathan LM et al. (2014). Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms. Environ Sci Technol 48, 12,800–12,806. PMID: 25299381

Norton SE et al. (2013). A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin Biochem 46, 1,561–1,565. PMID: 23769817

O’Bleness M et al. (2014). Finished sequence and assembly of the DUF1220-rich 1q21 region using a haploid human genome. BMC Genomics 15, 387. PMID: 24885025

Oellerich M et al. (2014). Use of graft-derived cell-free DNA as an organ integrity biomarker to reexamine effective tacrolimus trough concentrations after liver transplantation. Ther Drug Monit 36, 136–140. PMID: 24452066

Oxnard GR et al. (2014). Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin Cancer Res 20, 1,698–1,705. PMID: 24429876

Patton S et al. (2014). Assessing standardization of molecular testing for non-small-cell lung cancer: Results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing. Br J Cancer 111, 413–420. PMID: 24983368

Persaud D et al. (2013). Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 369, 1,828–1,835. PMID: 24152233

Pinheiro LB et al. (2012). Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84, 1,003–1,011. PMID: 22122760

Podlesniy P et al. (2013). Low cerebrospinal fluid concentration of mitochondrial DNA in preclinical Alzheimer disease. Ann Neurol 74, 655–668. PMID: 23794434

Poenitzsch Strong AM et al. (2014). microRNA-340 as a modulator of RAS-RAF-MAPK signaling in melanoma. Arch Biochem Biophys 563, 118–124. PMID: 25043973

Ponomarenko EA et al. (2014). Chromosome 18 transcriptoproteome of liver tissue and HepG2 cells and targeted proteome mapping in depleted plasma: Update 2013. J Proteome Res 13, 183–190. PMID: 24328317

Porensky PN et al. (2012). A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse. Hum Mol Genet 21, 1,625–1,638. PMID: 22186025

Pornprasert S and Prasing W (2014). Detection of alpha(0)-thalassemia South-East Asian-type deletion by droplet digital PCR. Eur J Haematol 92, 244–248. PMID: 24330258

Potrich C et al. (2014). OncomiR detection in circulating body fluids: A PDMS microdevice perspective. Lab Chip 14, 4,067–4,075. PMID: 25178053

Qin J et al. (2014). Preservation of circulating cell-free fetal RNA in maternal blood using a blood collection device containing a stabilizing reagent. J Mol Genet Med 8, 097.

Rački N et al. (2014). One-step RT-droplet digital PCR: A breakthrough in the quantification of waterborne RNA viruses. Anal Bioanal Chem 406, 661–667. PMID: 24276251

Rački N et al. (2014). Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil, and water samples. Plant Methods 10, 307. PMID: 25628753

Rebolledo-Jaramillo B et al. (2014). Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 111, 15,474–15,479. PMID: 25313049

Reid AL et al. (2014). Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR. Clin Biochem, Dec 16 [Epub ahead of print]. Accessed January 29, 2015. PMID: 25523300

Roberts CH et al. (2013). Development and evaluation of a next-generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections. J Clin Microbiol 51, 2,195–2,203. PMID: 23637300

Roberts CH et al. (2014). Killer-cell immunoglobulin-like receptor gene linkage and copy number variation analysis by droplet digital PCR. Genome Med 6, 20. PMID: 24597950

Robins HS et al. (2013). Digital genomic quantification of tumor-infiltrating lymphocytes. Sci Transl Med 5, 214ra169. PMID: 24307693

Rothrock MJ Jr et al. (2013). Quantification of zoonotic bacterial pathogens within commercial poultry processing water samples using droplet digital PCR. Advances in Microbiology 3, 403–411.

Rungrassamee W et al. (2014). Mannooligosaccharides from copra meal improves survival of the Pacific white shrimp (Litopenaeus vannamei) after exposure to Vibrio harveyi. Aquaculture 434, 403– 410.

Sanmamed MF et al. (2015). Quantitative cell-free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow-up of patients with melanoma being treated with BRAF inhibitors. Clin Chem 61, 297–304. PMID: 25411185

Schuler B et al. (2014). Endogenous a-calcitonin-gene-related peptide promotes exercise-induced, physiological heart hypertrophy in mice. Acta Physiol (Oxf.) 211, 107–121. PMID: 24479375

Sedlak RH and Jerome KR (2013). Viral diagnostics in the era of digital polymerase chain reaction. Diagn Microbiol Infect Dis 75, 1–4. PMID: 23182074

Sedlak RH et al. (2014). Clinical utility of droplet digital PCR for human cytomegalovirus. J Clin Microbiol 52, 2,844–2,848. PMID: 24871214

Sedlak RH et al. (2014). Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin Chem 60, 765–772. PMID: 24594780

Shlush LI et al. (2014). Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 506, 328–333. PMID: 24522528

Siravegna G and Bardelli A (2014). Minimal residual disease in breast cancer: In blood veritas. Clin Cancer Res 20, 2,505–2,507. PMID: 24658155

Stieglitz E et al. (2015). Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia. Blood 125, 516–524. PMID: 25395418

Strain MC and Richman DD (2013). New assays for monitoring residual HIV burden in effectively treated individuals. Curr Opin HIV AIDS 8, 106–110. PMID: 23314907

Strain MC et al. (2013). Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One 8, e55943. PMID: 23573183

Sun B et al. (2014). Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction. Biotechniques 56, 319–325. PMID: 24924392

Sze MA et al. (2014). A comparison between droplet digital and quantitative PCR in the analysis of bacterial 16S load in lung tissue samples from control and COPD GOLD 2. PLoS One 9, e110351. PMID: 25329701

Takahashi K et al. (2014). Analysis of extracellular RNA by digital PCR. Front Oncol 4, 129. PMID: 24926435

Takahashi K et al. (2014). Modulation of hypoxia-signaling pathways by extracellular linc-RoR. J Cell Sci 127, 1,585–1,594. PMID: 24463816

Tamayo E et al. (2014). Quantification of IgM molecular response by droplet digital PCR as a potential tool for the early diagnosis of sepsis. Crit Care 18, 433. PMID: 25043844

Taylor SD et al. (2014). Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain. Aging Cell 13, 29–38. PMID: 23911137

Tebas P et al. (2014). Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med 370, 901–910. PMID: 24597865

Usmani-Brown S et al. (2014). Analysis of b-cell death in type 1 diabetes by droplet digital PCR. Endocrinology 155, 3,694–3,698. PMID: 25004096

Vasilev V et al. (2014). McCune-Albright syndrome: A detailed pathological and genetic analysis of disease effects in an adult patient. J Clin Endocrinol Metab 99, E2,029–E2,038. PMID: 25062453

Vojtech L et al. (2014). Exosomes in human semen carry a distinctive repertoire of small non- coding RNAs with potential regulatory functions. Nucleic Acids Res 42, 7,290–7,304. PMID: 24838567

Wang C et al. (2014). Decreased HIV type 1 transcription in CCR5-Δ32 heterozygotes during suppressive antiretroviral therapy. J Infect Dis 210, 1,838–1,843. PMID: 24935955

Wang IX et al. (2013). ADAR regulates RNA editing, transcript stability, and gene expression. Cell Rep 5, 849–860. PMID: 24183664

Weber ND et al. (2014). AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication. PLoS One 9, e97579. PMID: 24827459

White RA III et al. (2013). Draft genome sequence of Exiguobacterium pavilionensis strain RW-2, with wide thermal, salinity, and pH tolerance, isolated from modern freshwater microbialites. Genome Announc 8, e00597–e00613. PMID: 23929485

White TB et al. (2014). A droplet digital PCR detection method for rare L1 insertions in tumors. Mobile DNA 5, 111. PMID: 25598847

Wiencke JK et al. (2014). A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood. Epigenetics 9, 1,360 –1,365. PMID: 25437051

Witwer KW et al. (2013). Real-time quantitative PCR and droplet digital PCR for plant miRNAs in mammalian blood provide little evidence for general uptake of dietary miRNAs: Limited evidence for general uptake of dietary plant xenomiRs. RNA Biol 10, 1,080–1,086. PMID: 23770773

Wong TN et al. (2014). Role of TP53 mutations in the origin and evolution of therapy- related acute myeloid leukaemia. Nature, Dec 8 [Epub ahead of print]. Accessed January 29, 2015. PMID: 25487151

Yamada T et al. (2013). EGFR T790M mutation as a possible target for immunotherapy; identification of HLA-A*0201-restricted T cell epitopes derived from the EGFR T790M mutation. PLoS One 8, e78389. PMID: 24223798

Yamasaki S et al. (2014). Generation of human induced pluripotent stem (lps) cells in serum- and feeder-free defined culture and TGF-b1 regulation of pluripotency. PLoS One 9, e87151. PMID: 24489856

Yang R et al. (2014). Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples. Int J Parasitol 44, 1,105–1,113. PMID: 25229177

Yukl SA et al. (2014). Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR. Biotechniques 56, 194–196. 24724845

Zhao H et al. (2013). Specific qPCR assays for the detection of orf virus, pseudocowpox virus, and bovine papular stomatitis virus. J Virol Methods 194, 229–234. PMID: 24035807

Zhu Y et al. (2014). XSAnno: A framework for building ortholog models in cross-species transcriptome comparisons. BMC Genomics 15, 343. PMID: 24884593

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