Droplet DigitalTM PCR Research Articles

A novel ddPCR™ assay for eDNA detection and quantification of Greater Amberjack Seriola dumerilli and three congeners in US waters: challenges and application to fisheries independent surveys

Background Four Seriola species support recreational and commercial fisheries along the U.S. Atlantic Ocean and the Gulf of Mexico, with the S. dumerili Gulf of Mexico stock being overfished for over three decades. The study presented here is part of a fisheries-independent project initiated to determine an absolute abundance of S. dumerili, to expand biological knowledge of the species and to develop novel tools for fisheries management. Environmental DNA (eDNA) tools aimed at the detection and quantification of target species are starting to emerge in support of marine fisheries surveys. Key to progressing the field is Droplet Digital™ PCR (ddPCR™), a highly sensitive technique with advanced multiplexing and direct quantification capabilities that can provide fisheries scientists with improved interpretation of eDNA data. Methods We developed and validated a novel tetraplex ddPCR™ assay able to detect and distinguish between S. dumerili, S. fasciata, S. rivoliana, and S. zonata from seawater eDNA samples. In order to groundtruth ddPCR™ data, and explore its capacity to provide abundance estimates, we compared ddPCR™ detections and quantifications to abundance data inferred from multiple camera (ROV, S-BRUV, chevron trap) and acoustic (VPS array) gears deployed during a fisheries research gear-calibration cruise. Results We demonstrated that with eDNA contamination controls and best practice protocols, it is viable to conduct eDNA research as part of a fisheries survey cruise. eDNA sampling was completed in less time than camera gears (15 min vs 2 h). Both eDNA and camera gears detected the presence of S. dumerili and S. rivoliana at both sites and all sampling days, but not S. fasciata and S. zonata. eDNA concentration data was higher for S. dumerili than S. rivoliana at both sites for all sampling days, in line with abundance patterns obtained from camera gears. The highest correlation (r = 0.97) was obtained between the measures of eDNA between gear deployments and ROV. Discussion Incorporating eDNA in fisheries surveys would not require additional days at sea and could improve precision in fish detection and abundance. eDNA can be a valuable complement to camera gears deployed in geographic areas or seasons with poor visibility conditions, where fish may be present but cannot be confidently identified to the species level. The high correlation obtained between ROV and eDNA data collected between gear deployments adds to a growing number of studies demonstrating the potential of eDNA as an indicator of abundance for fisheries stock assessments. Time-series data from a carefully designed eDNA survey, that estimates relative abundance, could be used as an index of relative abundance for the S. dumerili stock assessment. To achieve this, investment into follow-up studies with increased sample sizes and spatial and temporal replication would be necessary to allow for year-to-year comparisons and validate the robustness of the correlation observed.

Impact of water stress on <i>Phaeomoniella chlamydospora</i> abundance and Petri disease symptom development in young grapevines

Phaeomoniella chlamydospora (Pch) is one of the main pathogens causing Petri disease, a grapevine trunk disease responsible for the decline and mortality of grapevines within a few years after planting. Phaeomoniella chlamydospora has been shown to be prevalent in asymptomatic grapevine nursery material, leading to the hypothesis that it may act as a latent pathogen, transitioning from an endophytic to a pathogenic phase under grapevine stress. To investigate this hypothesis, a two-year greenhouse and a four-year field experiment were conducted on young self-rooted ‘Merlot’, and ‘Merlot’ grafted onto ‘SO4’ rootstock, artificially inoculated with different spore concentrations of Pch and subjected to water stress. Additionally, the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis was inoculated in the soil in the greenhouse experiment to investigate its effects on abiotic stress mitigation and thus, disease development in water stressed and non-stressed grapevines. DNA was extracted from the grapevine wood, and Droplet Digital™ PCR was conducted to determine Pch abundance before and after the experiments. In the greenhouse, Pch abundance in inoculated grapevines was greater in water stressed grapevines treated with AM than in stressed grapevines without AM or in non-stressed grapevines. Basal necrosis was greater in grapevines inoculated with Pch. In the field, Pch abundance was not affected by water stress, but basal necrosis was greater in grapevines inoculated with a high spore concentration of the fungus. Symptoms resembling Petri disease developed in the third year of the field experiment, where water stress increased grapevine mortality. This study shows that water stress may increase Pch abundance and mortality in young grapevines within the first few years after planting.

Abstract 6605: Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology

Abstract Introduction: Isolation and identification of circulating tumor cells (CTCs) offers a promising non-invasive diagnostic tool for cancer research, but significant technical hurdles have made practical application challenging. Recent studies have shown that CTCs undergo cellular changes, such as an epithelial-mesenchymal transition (EMT), with disease progression and metastasis, implying the heterogeneity of the tumor cell population in circulation. Systems for CTC isolation are often limited by a requirement for epithelial markers or separate systems for identification of CTCs. As such, an automated platform that successfully isolates both epithelial and mesenchymal cells and enables identification of cells is necessary for successful study of CTCs. The Genesis System from Bio-Rad™ Laboratories, Inc. isolates rare CTCs based on size and deformability. It also enables users to identify isolated cells through use of an on-slide immunofluorescence (IF) staining workflow, which minimizes sample manipulation and potential sample loss. Methods: In order to achieve high-throughput label-free CTC isolation, the Genesis System uses Celselect Slides™. Each slide contains 56,400 individual microchambers to capture CTCs while maintaining viability. This Celselect Slide technology can be used for either enrichment or enumeration protocols. For this study, enumeration protocols were performed to capture and identify CTCs using on-slide IF staining. Enrichment protocols were utilized to capture, enrich, and retrieve CTCs for downstream applications. The performance was evaluated using a breast cancer cell line, MCF-7, spiked into whole blood from healthy donors. Results: The slide isolated 83.95 ± 10.25% of spiked cancer cells in blood samples at a concentration of 10-250 cells/ml. During the CTC enrichment procedure, 99.92% of blood cells were eliminated. Afterwards, 70% of the isolated CTCs were successfully released from the slide and over 92% of the released cells remained viable. Discussion and Conclusion: We present a fully automated platform to isolate and identify CTCs using on-slide immunofluorescence staining, which minimizes inevitable cell loss during the workflow. Captured cells can be retrieved for downstream applications to facilitate discovery of unknown CTC functions and their clinical relevance in cancer research. The wide range of possible downstream applications include Droplet Digital™ PCR, quantitative PCR, western blot assays, bulk/single-cell genomics, cell culture, fluorescence in situ hybridization, and RNA-Seq. Additionally, the design of the system is amenable to other biofluids which may be utilized for liquid biopsies. The Genesis System with Celselect Slide technology offers a powerful diagnostic tool to monitor disease progression and personalized treatment. Citation Format: Yoon-Tae Kang, Errile Pusod, Ally Crudgington, Abiodun Bodunrin, Nick Bosio, Susana Chan, David Coe, Michelle Racey. Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6605.

Comparison of environmental microbiomes in an antibiotic resistance-polluted urban river highlights periphyton and fish gut communities as reservoirs of concern

Natural waterways near urban areas are heavily impacted by anthropogenic activities, including their microbial communities. A contaminant of growing public health concern in rivers is antibiotic resistant genes (ARGs), which can spread between neighboring bacteria and increase the potential for transmission of AR bacteria to animals and humans. To identify the matrices of most concern for AR, we compared ARG burdens and microbial community structures between sample types from the Scioto River Watershed, Ohio, the United States, from 2017 to 2018. Five environmental matrices (water, sediment, periphyton, detritus, and fish gut) were collected from 26 river sites. Due to our focus on clinically relevant ARGs, three carbapenem resistance genes (blaKPC, blaNDM, and blaOXA-48) were quantified via DropletDigital™ PCR. At a subset of nine urbanized sites, we conducted16S rRNA gene sequencing and functional gene predictions. Carbapenem resistance genes were quantified from all matrices, with blaKPC being the most detected (88 % of samples), followed by blaNDM (64 %) and blaOXA-48 (23 %). Fish gut samples showed higher concentrations of blaKPC and blaNDM than any other matrix, indicating potential ARG bioaccumulation, and risk of broader dissemination through aquatic and nearshore food webs. Periphyton had higher concentrations of blaNDM than water, sediment, or detritus. Microbial community analysis identified differences by sample type in community diversity and structure. Sediment samples had the most diverse microbial communities, and detritus, the least. Spearman correlations did not reveal significant relationships between the concentrations of the monitored ARGs and microbial community diversity. However, several differentially abundant taxa and microbial functions were identified by sample type that is definitive of these matrices' roles in the river ecosystem and habitat type. In summary, the fish gut and periphyton are a concern as AR reservoirs due to their relatively high concentration of carbapenem resistance genes, diverse microbial communities, and natural functions that promote AR.

Development of a Droplet Digital™ PCR DNA methylation detection and quantification assay of prenatal tobacco exposure.

DNA methylation is a labile modification associated with gene expression control and environmental adaptations. High throughput, scalable and quantitative assessments of specific DNA methylation modifications in complex genomic regions for use in large population studies are needed. The performance of Droplet Digital™ PCR (ddPCR™) was investigatedfor DNA methylation detection against next-generation bisulfite sequencing (NGS) to demonstrate the ability of ddPCRto detect and validate DNA methylation levels and complex patterns among neighboring CpGs in regions associated with prenatal tobacco exposure. While both techniques are reproducible, ddPCRdemonstrates a unique advantage for high-throughput DNA methylation analysis in large-scale population studies and provides the specificity to accurately measure DNA methylation of target CpGs in complex regions.

PSX-B-15 Real-time quantitative PCR versus droplet digital PCR for measurement of peripheral blood leukocytes interferon-tau stimulated genes in beef cattle

Abstract We aimed to compare the abundance of interferon-tau stimulated genes (ISG) transcripts in peripheral blood leukocytes of artificially inseminated beef cows using real-time quantitative PCR (RT-qPCR) versus Droplet Digital PCR (ddPCR). Multiparous Bos taurus beef cows (n = 7) were subjected to timed artificial insemination (TAI) on day 0. Pregnancy was determined by transrectal ultrasonography on days 26 and 30 post-TAI, and cows were classified as: pregnant (n=4; embryo detected on days 26 and 30) or non-pregnant (n = 3; no embryo detected). Coccygeal vein blood samples were collected on days 0, 15, 17, 19, 20 and 24 post-TAI. Leukocyte RNA was extracted from the buffy coat fraction using Trizol associated with the DirectZol-RNA kit and transcribed to cDNA. The abundance of ISG (ISG15 and MX2) was assessed by relative quantification to a reference gene (RPS9) using RT-qPCR and by absolute quantification using the QX100TM Droplet DigitalTM PCR System (Bio-rad Laboratories) according to manufacturer’s recommendations. Data were analyzed using PROC MIXED on SAS 9.4. For the RT-qPCR, pregnant cows had greater (P < 0.05) ISG15 and MX2 abundance compared to non-pregnant cows on days 20 (ISG15:0.11±0.1 vs. 0.01±0.001 and MX2:0.73±0.4 vs. 0.06±0.06) and 24 (ISG15:0.34±0.2 vs. 0.01±0.001 and MX2:0.77±0.2 vs. 0.13±0.04). For ddPCR, a greater ISG15 and MX2 copy numbers in pregnant cows was observed on days 15 (ISG15:129 vs. 44 copies/µl and MX2:33 vs. 10 copies/µl) and 20 (ISG15: 216 vs. 30 copies/µl and MX2: 44 vs. 7 copies/µl), and also on day 24 for ISG15 (32 vs. 7 copies/µl) compared to non-pregnant cows. In conclusion, ddPCR was able to detect an earlier expression of ISG in pregnant cows. Future studies are needed to enroll more animals and establish a suitable cutoff value using ddPCR, which could be less subjective for diagnosis as it does not require the use of a reference gene.

DNA recovery from Droplet Digital™ PCR emulsions using liquid nitrogen.

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.

A Novel Description of the Human Sinus Archaeome During Health and Chronic Rhinosinusitis.

Human microbiome studies remain focused on bacteria, as they comprise the dominant component of the microbiota. Recent advances in sequencing technology and optimization of amplicon sequencing protocols have allowed the description of other members of the microbiome, including eukaryotes (fungi) and, most recently, archaea. There are no known human-associated archaeal pathogens. Their diversity and contribution to health and chronic respiratory diseases, such as chronic rhinosinusitis (CRS), are unknown. Patients with CRS suffer from long-term sinus infections, and while the microbiota is hypothesized to play a role in its pathogenesis, the exact mechanism is poorly understood. In this cross-sectional study, we applied a recently optimized protocol to describe the prevalence, diversity and abundance of archaea in swab samples from the middle meatus of 60 individuals with and without CRS. A nested PCR approach was used to amplify the archaeal 16S rRNA gene for sequencing, and bacterial and archaeal load (also based on 16S rRNA genes) were estimated using Droplet Digital™ PCR (ddPCR). A total of 16 archaeal amplicon sequence variants (ASVs) from the phyla Euryarchaeota and Thaumarchaeota were identified. Archaeal ASVs were detected in 7/60 individuals, independent of disease state, whereas bacterial ASVs were detected in 60/60. Bacteria were also significantly more abundant than archaea. The ddPCR method was more sensitive than amplicon sequencing at detecting archaeal DNA in samples. Phylogenetic trees were constructed to visualize the evolutionary relationships between archaeal ASVs, isolates and clones. ASVs were placed into phylogenetic clades containing an apparent paucity of human-associated reference sequences, revealing how little studied the human archaeome is. This is the largest study to date to examine the human respiratory-associated archaeome, and provides the first insights into the prevalence, diversity and abundance of archaea in the human sinuses.

Analysis of cancer-related mutations in extracellular vesicles RNA by Droplet Digital™ PCR.

Extracellular vesicles (EVs) are taking their place as potential biomarkers in the field of liquid biopsy. In this study, EVs were isolated from plasma samples of 31 patients with colorectal cancer and melanoma via differential centrifugation and Droplet Digital™ PCR (Bio-Rad, CA, USA) was used to profile BRAF V600E/K, KRAS G12A/C/D/V and KRASG13D mutations from EV-derived cDNA. The concordance rates with corresponding tissue were 54%and 44% in the colorectal cancer and melanoma cohort, respectively. Two patients displayed mutations in EVs not previously detected in tissue as evidence for emerging molecular resistance to anti-EGFR and BRAF/MEK inhibitor therapy prior to radiological evidence of tumor progression. We concluded that EV-derived nucleic acids may provide clinically relevant diagnostic information and mirror evolution of the disease.

An environmental DNA tool for monitoring the status of the Critically Endangered Smalltooth Sawfish, Pristis pectinata, in the western Atlantic

The Critically Endangered Smalltooth Sawfish, Pristis pectinata, was once widespread in the tropical and subtropical waters of the Atlantic Ocean, but following substantial declines over the past century, the remaining population(s) are currently confined to Florida in the U.S., and the Bahamas. Recent research and verified public encounter reports suggest that the core population in south and southwest Florida may be stabilizing and potentially expanding into formerly occupied areas of their historic range in the western Atlantic; however, the status of this species outside of core waters is not well understood. Environmental DNA (eDNA) methods provide a relatively cost effective and rapid assessment tool for monitoring species occurrence in aquatic habitats. Here, we have developed an eDNA tool: a species-specific Droplet Digital™ PCR assay targeting a 100-base pair portion of the mitochondrial NADH dehydrogenase subunit 2 gene in P. pectinata, with the ability to reliably detect as little as 0.25 pg of target DNA. The assay was validated by analyzing a water sample from an occupied nursery in southwest Florida, which was found to contain an average of 11.54 copies of target DNA/µL (SE = 0.72) in the reaction. The assay was then further tested by placing a juvenile sawfish in an ex situ tank and analyzing water samples collected at time intervals. The implementation of this eDNA tool into field surveys will provide additional, reliable data to assess species recovery and aid in prioritizing localities in which to focus new research, conservation, and education initiatives.

Abstract P5-11-12: Clinical value of ESR1 mutations from cell free DNA in ethynil estradiol treatment for metastatic breast cancer

Abstract Background: There are some cases in which estrogen addition therapy with ethinyl estradiol (EE2) is effective in cases in which aromatase inhibotor (AI) acquired resistance has been achieved after prolonged administration. In such case, ESR1 mutation was reported to be frequently seen. In this study, we examined the relation between histopathological factors, or ESR1 mutations detected by multiplex droplet digital PCR from cell free DNA (cfDNA) in patients’ and the effect of EE2 treatment. Patients and methods: From 2011 to 2016, we conducted a prospective observational study of EE2 therapy (0.5 to 3 mg / day PO) in our department (registration No. UMIN000002831). ER/PgR expression by IHC in a part cases, and serum E2 and FSH levels and blood cell counts in all cases were evaluated before/after treatment. Droplet digital PCR (ddPCR) system by the QX200™ Droplet Digital™ PCR System (Bio-Rad laboratories, Hercules, CA, USA) which makes thousands of droplets and each of them contained one or no copy of the sequence of ESR1 gene reacted with a pair of primers and two TaqMan probes. The PCR data were quantified using QuantaSoft™ software (Bio-Rad laboratories) and data are expressed as a percentage of mutant to total (mutant plus wild type) for each sample. Results: In this series, partial response (PR) was seen in 30% (12/40), long stable disease 12.5% (5/40), clinical benefit rate (CBR) 42.5% (17/40). The median EE2 dosing period in cases with CB was 8.4 months. AI was re-administered to 17 cases for which efficacy was obtained by EE2, and 8 cases had CB. After that, EE2 was re-challenge to 4 cases obtained effective cases. After EE2 treatment, serum E2 level increased and FSH decreased. In addition, the neutrophil-lymphocyte ratio increased, but its biological significance was unknown. Point mutations (Y537S, Y537N, D538G) of the ESR1 gene in cell free DNA in plasma were searched in 21 cases before EE2 administration, and High frequent ESR1 mutations were seen in 10 cases (47%) in any mutative site. In the patients with wild type of ESR1 before treatment, there were tended to be more effective for EE2 treatment, but they were not statistically significant. Additionally, no relationship between PIK3CA mutations (E542x, E545x, H1047x, G1049x) and EE2 effect was seen. Conclusion: The action mechanisms of EE2 treatment were reported as estrogen-induced apoptosis, which may not be related to ESR1 nor PIK3CA mutations. Further research related to EE2 treatment with biologic factors, such as gene alterations of the related factors, must be required. Citation Format: Hirotaka Iwase, Takashi Takeshita, Mutsuko Ibusuki, Aiko Sueta, Mai Tomiguchi, Yoshitaka Fujiki, Yutaka Yamamoto. Clinical value of ESR1 mutations from cell free DNA in ethynil estradiol treatment for metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-11-12.

Digital droplet PCR and IDAA for the detection of CRISPR indel edits in the malaria species Anopheles stephensi.

CRISPR/Cas9 technology is a powerful tool for the design of gene-drive systems to control and/or modify mosquito vector populations; however, CRISPR/Cas9-mediated nonhomologous end joining mutations can have an important impact on generating alleles resistant to the drive and thus on drive efficiency. We demonstrate and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi: Indel Detection by Amplicon Analysis (IDAA™) and Droplet Digital™ PCR (ddPCR™). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis in cage trials and contained field testing of gene-drive mosquitoes.

T790M mutations identified by circulating tumor DNA test in lung adenocarcinoma patients who progressed on first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

Background:Plasma circulating tumor deoxyribonucleic acid (ctDNA) test is an alternative method to detect the T790M mutation. Compared to conventional tumor rebiopsy, ctDNA possesses several advantages including less invasive, faster, lower costs, and having minimal risk of complications for patients.Objective:The main objective of the study is to identify the prevalence of T790M mutations in lung adenocarcinoma patients who progressed after tyrosine kinase inhibitors (TKIs) therapy using ctDNA examination.Materials and Methods:This was a retrospective cohort study based on medical records of lung adenocarcinoma patients in the Oncology Outpatient Clinic of Dr. Soetomo General Hospital within the period of January 2017–June 2018. Patients who progressed after receiving first-line epidermal growth factor receptor-TKI (EGFR-TKI) undergone plasma ctDNA examination and genotyping using digital platforms (Droplet Digital™ PCR) method.Results:In total, there were 39 patients who met the criteria for ctDNA testing. Thirty-three patients (84.6%) received first-line gefitinib, while the other six (15.4%) received erlotinib. The T790M mutations were detected in 46.2% of patients. In addition, EGFR common mutation in exon 19 and exon 21 were detected in 87.2% of patients. Median progression-free survival of patients receiving first-line gefitinib or erlotinib were both around 9 months and did not differ significantly.Conclusions:CtDNA examination successfully detected T790M mutation in a certain proportion of lung adenocarcinoma patients who progressed after first-line EGFR-TKI without the need for difficult and invasive rebiopsy.

Development of highly sensitive environmental DNA methods for the detection of Bull Sharks, Carcharhinus leucas (Müller and Henle, 1839), using Droplet Digital™ PCR

AbstractBackgroundAs apex and mesopredators, elasmobranchs play a crucial role in maintaining ecosystem function and balance in marine systems. Elasmobranch populations worldwide are in decline as a result of exploitation via direct and indirect fisheries mortalities and habitat degradation; however, a lack of information on distribution, abundance, and population biology for most species hinders their effective management. Environmental DNA analysis has emerged as a cost‐effective and non‐invasive technique to fill some of these data gaps, but often requires the development of species‐specific methodologies.AimsHere, we established eDNA methodology appropriate for targeted species detections of Bull Sharks, Carcharhinus leucas, in estuarine waters in the northern Gulf of Mexico.Materials and MethodsWe compared different QIAGEN®DNeasy® extraction kit protocols and developed a species‐specific Droplet Digital™ PCR (ddPCR) assay by designing primers and an internal probe to amplify a 237 base pair portion of the ND2 gene in the mitochondrial genome of C. leucas. To validate the developed methods, water samples were collected from known C. leucas habitat and from an ex situ closed environment containing a single C. leucas individual. The effectiveness of the assay in an open environment was then assessed by placing one C. leucas into a flow‐through mesocosm system and water samples were collected every 30 min for 3 hr.ResultsThe developed C. leucas ‐specific assay has the ability to detect target DNA concentrations in a reaction as low as 0.6 copies/μl. DdPCR reactions performed on water samples from known habitat and 30 min after a shark was added to the closed environment contained 1.62 copies/μl and 166.6 copies/μl of target C. leucas eDNA, respectively. Carcharhinus leucas eDNA was detected in the flow‐through system within 30 min, but concentrations remained low and variable throughout the duration of the experiment.

Recurrent fires do not affect the abundance of soil fungi in a frequently burned pine savanna.

While the negative effects of infrequent, high-intensity fire on soil fungal abundance are well-understood, it remains unclear how the short-term history of frequent, low-intensity fire in fire-dependent ecosystems impacts abundance, and whether this history governs any abundance declines. We used prescribed fire to experimentally alter the short-term fire history of patches within a fire-frequented old-growth pine savanna over a 3 y period. We then quantified fungal abundance before and after the final fire using phospholipid fatty acid (PLFA) assays and Droplet Digital™ PCR (ddPCR). Short-term fire history largely did not affect total fungal abundance nor pre- to post-fire abundance shifts. While producing similar conclusions, PLFA and ddPCR data were not correlated. In addition to piloting a new method to quantify soil fungal abundance, our findings indicate that, within fire-frequented pine savannas, recurrent fires do not consistently decrease total fungal abundance, and abundance changes are not contingent upon short-term fire history. This suggests that many fungi in fire-dependent ecosystems are fire-tolerant.

Abstract 5139: Development of novel copy number variation (CNV) reference materials in genome in a Bottle background

Abstract Introduction: Advancements in tumor characterization by solid and liquid biopsy are instrumental in the evolution of personalized medicine. Recent improvements in assay sensitivity for profiling DNA variants such as single nucleotide polymorphisms (SNP) and indels have facilitated expansion of both solid and liquid biopsy applications. However, measurement of copy number variations (CNV) is a relatively new application, and few reference materials exist to aid in assay development and optimization. In this study, we demonstrate for the first time the development of 17 whole gene CNV reference standards in a background of the highly characterized NIST Genome in a Bottle GM24385 genomic DNA. Method: DNA molecules containing full genes of MET, ERBB2, ERBB3, PIK3CA, EGFR, BRAF, FGFR1, FGFR2, FGFR4, KIT, KRAS, MYC-N, PDGFRA, MDM2, CD274, MYC-L and MYC were each spiked separately into background GM24385 genomic DNA to generate 2.8 copies of each gene. Copy number for each of the 17 CNV samples was determined using Bio-Rad® Droplet DigitalTM PCR (ddPCRTM). To assess linearity over a range of copy numbers, MET and ERBB2 CNV ladder reference materials with copy gains at 6 different levels 3, 6, 9, 12 and 15 copies were also generated. To mimic circulating tumor DNA, each CNV sample was fragmented and size selected to recover a population containing ~170 bp size fragments. Copy number was also verified post size selection using ddPCRTM and the Ion S5TM XL system. Multiplexed triple CNV controls with MET, EFGR and ERBB2 were also developed at 3, 6, 9, 12 and 15 copies and analyzed by ddPCRTM. Results: Copy number for all 17 genes measured at 2.80 ± 0.28 copies by ddPCR. When tested in a ctDNA copy number ladder format, MET and ERBB2 showed good linearity of R2=0.99 for observed versus expected # of copies. Good correlation between ddPCR and NGS was also observed, with a slope of 1.004 for MET and 0.969 for ERBB2 across increasing copy numbers and standard deviation (SD) of ± 10%. Notably, SD increased at copy numbers higher than 12. The average size of fragmented ctDNA CNV reference standards were observed to be ~170 bp on the Agilent 2100 Bioanalyzer system. Finally, the multiplexed triple CNV control also performed as expected on ddPCR with SD of ± 10%. Conclusion: A novel method for producing CNV and ctDNA CNV controls has been developed that enables preparation of any copy gain level with a known gDNA background. Simpler QC materials mimicking patient samples will enable simpler CNV test method development and analytical validation, which will be critical for laboratories to introduce CNV solid and liquid biopsy testing into the field. Citation Format: Kunal Banjara, Lu Zheng, Samyuktha Dasari, Kara Norman. Development of novel copy number variation (CNV) reference materials in genome in a Bottle background [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5139.

MiRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses.

This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan® technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR® Green is not used but the more sensitive TaqMan® probe that on 5' end contains a covalently attached fluorophore and on 3' quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.

Abstract 5574: A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples

Abstract Introduction: Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We describe a method that combines a kittable and efficient wet-bench workflow with accurate dry-bench analytics to reduce costs and turnaround time, and is relevant to clinical research and patient testing. Methods: We developed a comprehensive, targeted NGS technology to enable the ultra-sensitive detection of variants from liquid biopsy samples. Input DNA molecules from Seraseq™ ctDNA v2 reference materials (SeraCare) and >50 matched FFPE and plasma samples were uniquely tagged with a random molecular barcode (MBC), amplified in an efficient PCR protocol, and sequenced using a targeted panel covering >500 somatic mutation hotspots. Sequence-ready libraries were prepared from input DNA within 9 hours. Data were analyzed with a robust bioinformatics pipeline tailored to the library chemistry to correct for multiple background errors. Variants were identified with a site-specific model, which effectively eliminated recurring non-biological aberrations that remained despite MBC-facilitated error-suppression. We verified variant calls in plasma by Droplet Digital™ PCR (Bio-Rad). To evaluate the limit of detection, healthy control and mutation-positive plasma admixtures were prepared and sequenced. Results: Input template molecules were efficiently recovered. The median number of unique MBCs across all amplicons was >90% of expectation based on input DNA quantities for both the tumor-associated plasma and Seraseq™ ctDNA v2 material. SNVs and indels were recognized with >90% sensitivity and PPV at 0.5% allele frequency (AF) in the Seraseq™ ctDNA mutation mixes. In both neat plasma samples and plasma admixtures, we correctly identified tumor mutations down to ~0.1% AF while maintaining a low false-positive rate (sensitivity and PPV remained high for AF ≥0.1%). Reference ctDNA material and tumor-associated plasma had analogous template amplification and variant frequency rates. Conclusions: A fast, efficient, and sensitive NGS panel approach was developed and evaluated, and it demonstrated the reliable and specific detection of rare variants in liquid biopsy specimens. The method is able to distinguish a low frequency ctDNA signal from the overwhelming background noise in plasma cell-free DNA using a streamlined workflow and purpose-built bioinformatics pipeline. This technology may provide an easy-to-use, high-performance, and adaptable NGS diagnostic framework for disease detection and therapeutic intervention monitoring. Citation Format: Jessica L. Larson, Liangjing Chen, Lando Ringel, Blake Printy, Farol L. Tomson, Yves Konigshofer, Sarah Statt, Joseph K. Kaplan, Shobha Gokul, Jeffrey Shelton, Gary J. Latham, Brian C. Haynes. A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5574.

Performance verification of a plasma-based PD-L1 test that reliably measures mRNA expression from patients with NCSLC.

156 Background: The detection of circulating nucleic acids using non-invasive blood-draws has become highly relevant to clinical testing. In this study we report on the development of a blood-based PD-L1 test for immunotherapy selection. Methods: We have previously reported on the analytic performance of a droplet digital™ PCR (ddPCR) assay for circulating cytokeratin 19 and PD-L1. Using a variable threshold based on a logistic regression score for the blood assay and a 1% IHC (immunohistochemistry) tissue cut-off, concordance was 80% (n = 16). Positive calls for the blood-based PD-L1 assay ranged from 2 to 124 copies. In this study we focused on variables that could impact concordance of the blood assay and tissue results. Criteria included droplet counts for tissue and blood mRNA transcripts and tumor proportion score (TPS) for the tissue IHC assay (22C3 pharmDx). Results: We examined the correlation between PD-L1 in formalin-fixed paraffin-embedded (FFPE) tissue by IHC, mRNA expression in serial cut FFPE sections, and in matched plasma samples collected at the time of tissue resection. Five cases were assayed to confirm PD-L1 positivity by tissue. We successfully recovered RNA from serial tissue sections for each case and detected PD-L1 levels ranging from 6 to 1272 copies. Plasma samples were available for four of the cases for circulating RNA evaluation, and we were successful in detecting PD-L1 in all cases (copy range 32-138). While all four cases contained detectible PD-L1 mRNA in tissue and circulation, we observed little concordance between these levels in tissue and blood. Conclusions: We have developed methods to measure the dynamic range of PD-L1 from plasma. We have shown feasibility of these methods by evaluating key immune and cancer-specific RNAs. The current study demonstrates that although the development of quantitative assays for mRNA in blood is possible, concordance with traditional clinical tissue assays such as IHC may not be a useful validation measure. We have initiated prospective validation studies that will continue to evaluate PD-L1 expression by IHC and in the blood-based assay, and will also monitor patient performance measures in response to immunotherapy.

Monitoring of Response and Resistance in Plasma of EGFR-Mutant Lung Cancer Using Droplet Digital PCR.

The identification of oncogenic driver mutations has led to the rapid rise of genotype-directed treatments. However, genetic analysis of tumors remains cumbersome and a morbid experience for patients. Noninvasive assessment of tumor genotype, so-called "liquid biopsy," such as plasma genotyping represents a potentially transformative tool. Here we describe a genotyping protocol of cell-free plasma DNA (cfDNA) using Droplet Digital™ PCR (ddPCR™). ddPCR emulsifies DNA into ~20,000 droplets in which PCR is performed to endpoint in each droplet for both mutant and wild-type DNA. Droplets are run through a modified flow cytometer where mutant and wild-type DNA emit different colored signals. The count of these signals upon Poisson distribution analysis allows sensitive quantification of allelic prevalence.