Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies

Abstract BACKGROUND Although interest in droplet-digital PCR technology (ddPCR) for cell-free circulating DNA (cfDNA) analysis is burgeoning, the technology is compromised by subsampling errors and the few clinical targets that can be analyzed from limited input DNA. The paucity of starting material acts as a “glass ceiling” in liquid biopsies because, irrespective how analytically sensitive ddPCR techniques are, detection limits cannot be improved past DNA input limitations. METHODS We applied denaturation-enhanced ddPCR (dddPCR) using fragmented genomic DNA (gDNA) with defined mutations. We then tested dddPCR on cfDNA from volunteers and patients with cancer for commonly-used mutations. gDNA and cfDNA were tested with and without end repair before denaturation and digital PCR. RESULTS By applying complete denaturation of double-stranded DNA before ddPCR droplet formation the number of positive droplets increased. dddPCR using gDNA resulted in a 1.9–2.0-fold increase in data-positive droplets, whereas dddPCR applied on highly-fragmented cfDNA resulted in a 1.6–1.7-fold increase. End repair of cfDNA before denaturation enabled cfDNA to display a 1.9–2.0-fold increase in data-positive signals, similar to gDNA. Doubling of data-positive droplets doubled the number of potential ddPCR assays that could be conducted from a given DNA input and improved ddPCR precision for cfDNA mutation detection. CONCLUSIONS dddPCR is a simple and useful modification in ddPCR that enables extraction of more information from low-input clinical samples with minor change in protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis in cancer and prenatal screening.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

10.21203/rs.3.rs-103611/v2 ◽

2021 ◽

Author(s):

Suttipat Srisut ◽

Kanokon Suwannasin ◽

Rungirun Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Antimalarial Drug Resistance ◽

Quantification Limit ◽

Field Samples ◽

The Cost

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: There were no significant differences between the GCN results obtained from uniplex andmultiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

10.21203/rs.3.rs-103611/v1 ◽

2020 ◽

Author(s):

Suttipat Srisut ◽

Kanokon Suwannasin ◽

Rungirun Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Antimalarial Drug Resistance ◽

Quantification Limit ◽

Field Samples

Abstract Background: Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum pfplasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with antimalarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to antimalarial drugs.Methods: The ddPCR assays were developed to detect the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.Results: Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. The accuracy and %RSD value of the duplex ddPCR assay were 94.88% and 3.71, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). Conclusions: The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of antimalarial drug resistance.

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Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method

PLoS ONE ◽

10.1371/journal.pone.0146784 ◽

2016 ◽

Vol 11 (1) ◽

pp. e0146784 ◽

Cited By ~ 11

Author(s):

Yanni Zhang ◽

En-Tzu Tang ◽

Zhiqiang Du

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Pcr Method

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Measurement of gene amplifications related to drug resistance in Plasmodium falciparum using droplet digital PCR

Malaria Journal ◽

10.1186/s12936-021-03659-5 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Suttipat Srisutham ◽

Kanokon Suwannasin ◽

Rungniran Sugaram ◽

Arjen M. Dondorp ◽

Mallika Imwong

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Copy Number ◽

Gene Copy Number ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Quantification Limit ◽

Field Samples ◽

The Cost

Abstract Background Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs. Methods A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay. Results There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated. Conclusions The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.

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Single-Nucleotide Polymorphism Leading to False Allelic Fraction by Droplet Digital PCR

Clinical Chemistry ◽

10.1373/clinchem.2017.273177 ◽

2017 ◽

Vol 63 (8) ◽

pp. 1370-1376 ◽

Cited By ~ 2

Author(s):

Eric S Christenson ◽

W Brian Dalton ◽

David Chu ◽

Ian Waters ◽

Karen Cravero ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Careful Analysis ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Clinical Samples ◽

Unexpected Finding ◽

Wild Type ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Patients With Cancer

Abstract BACKGROUND Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles. Clinical samples from patients with cancer with known SF3B1 mutations were collected and tested to evaluate the assays' ability to detect SF3B1 mutations in ptDNA. RESULTS Patient samples showed SF3B1 K700E mutations within the ptDNA of 4 patients with acute leukemia and 3 with myelodysplastic syndrome who were known to harbor this mutation. A blood sample from a patient with lung cancer with a known SF3B1 V701F mutation was also analyzed and this mutation was successfully identified in ptDNA. However, 1 of the patients with a K700E mutation was found to have a mutational burden of 98%. After careful analysis of this locus by Sanger sequencing and ddPCR, this patient was found to have an SNP (R702R), which prevented binding of the ddPCR wild-type probe to its cognate allele. CONCLUSIONS These results further support that ddPCR-based assays may be valuable companion diagnostics for the identification and monitoring of patients with cancer, but the results also emphasize the need to identify SNPs at loci that are being analyzed.

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Comparison of the Amplification Refractory Mutation System, Super Amplification Refractory Mutation System, and Droplet Digital PCR for T790 M Mutation Detection in Non-small Cell Lung Cancer after Failure of Tyrosine Kinase Inhibitor Treatment

Pathology & Oncology Research ◽

10.1007/s12253-017-0286-3 ◽

2017 ◽

Vol 24 (4) ◽

pp. 843-851 ◽

Cited By ~ 4

Author(s):

Lucheng Zhu ◽

Shirong Zhang ◽

Yanping Xun ◽

Yanping Jiang ◽

Bing Xia ◽

...

Keyword(s):

Lung Cancer ◽

Mutation Detection ◽

Kinase Inhibitor ◽

Digital Pcr ◽

Small Cell ◽

Droplet Digital Pcr ◽

Amplification Refractory Mutation System ◽

Small Cell Lung ◽

Mutation System ◽

Inhibitor Treatment

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The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

Hematological Oncology ◽

10.1002/hon.2489 ◽

2017 ◽

Vol 36 (2) ◽

pp. 429-435 ◽

Cited By ~ 31

Author(s):

Laura S. Hiemcke-Jiwa ◽

Monique C. Minnema ◽

Joyce H. Radersma-van Loon ◽

N. Mehdi Jiwa ◽

Mirthe de Boer ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Liquid Biopsies ◽

Sensitive Technique ◽

Highly Sensitive

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Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽

10.1161/hyp.68.suppl_1.p613 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Ryousuke Satou ◽

Akemi Katsurada ◽

Kayoko Miyata ◽

Andrei Derbenev ◽

Andrea Zsombok

Keyword(s):

Copy Number ◽

System Analysis ◽

Renin Angiotensin System ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Gene Copy ◽

Angiotensin System ◽

Renin Angiotensin ◽

Copy Numbers ◽

Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

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Darwin and evolutionary tales in leukemia

Hematology ◽

10.1182/asheducation-2009.1.3 ◽

2009 ◽

Vol 2009 (1) ◽

pp. 3-12 ◽

Cited By ~ 39

Author(s):

Mel Greaves

Keyword(s):

Charles Darwin ◽

Single Cell Analysis ◽

Lymphoblastic Leukemia ◽

Gene Copy Number ◽

Monozygotic Twins ◽

Chromosome Translocation ◽

Gene Copy ◽

Clinical Samples ◽

Evolutionary Divergence ◽

Evolutionary Pattern

AbstractAll cancers evolve by a process of genetic diversification and “natural selection” akin to the process first described by Charles Darwin for species evolution. The evolutionary, natural history of childhood acute lymphoblastic leukemia (ALL) is almost entirely covert, clinically silent and well advanced by the point of diagnosis. It has, however, been possible to backtrack this process by molecular scrutiny of appropriate clinical samples: (i) leukemic clones in monozygotic twins that are either concordant or discordant for ALL; (ii) archived neonatal blood spots or Guthrie cards from individuals who later developed leukemia; and (iii) stored, viable cord blood cells. These studies indicate prenatal initiation of leukemia by chromosome translocation and gene fusion (or hyperdiploidy) and the post-natal acquisition of multiple, gene copy number alterations (CNAs), mostly deletions. The prenatal or first “hit” occurs very commonly, exceeding the clinical rate of ALL by some 100× and indicating a low rate of penetrance or evolutionary progression. The acquisition of the critical, secondary CNAs requires some Darwinian selective advantage to expand numbers of cells at risk, and the cytokine TGF beta is able to exercise this function. The clonal architecture of ALL has been investigated by single cell analysis with multicolor probes to mutant genes. The data reveal not a linear sequence of mutation acquisition with clonal succession but rather considerable complexity with a tree-like or branching structure of genetically distinct subclones very reminiscent of Darwin’s original 1837 evolutionary divergence diagram. This evolutionary pattern has important implications for stem cells in ALL, for the origins of relapse and for therapeutic targeting.

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