Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses.

BACKGROUND: Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. METHODS: The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood-saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. RESULTS: The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood-saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030-0.9998 for SNVs and between 0.8883-0.9991 for small-indels in the case of the WGS protocol, and between 0.8643-0.999 for SNVs and between 0.7781-1.000 for small-indels in the case of the WES protocol. CONCLUSION: Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used.

Analysis of microRNAs in Small Urinary Extracellular Vesicles and Their Potential Roles in Pathogenesis of Renal ANCA-Associated Vasculitis.

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis. We used next-generation sequencing in the first preliminary study to detect the miRNAs with altered expression in uEVs of patients with AAV in comparison with age-matched controls. We confirmed the results using single-target quantitative polymerase chain reaction tests on different sets of samples and found five miRNAs (miR-30a-5p, miR-31-3p, miR-99a-5p, miR-106b-5p, miR-182-5p) with highly elevated levels in uEVs of patients. We performed the comparison of their targets with the differentially expressed proteins in uEVs of patients included in the first phase. We realized that upregulated miRNAs and proteins in uEVs in AAV patients target different biological pathways. The only overlap was detected in pathways regulating the actin cytoskeleton assembly and thus potentially affecting the glomerular functions. The associations of upregulated miRNAs with pathways that were neglected as components of complex AAV pathogenesis, e.g., the epidermal growth factor receptor signaling pathway, were found.

Improving structural variant clustering to reduce the negative effect of the breakpoint uncertainty problem.

BACKGROUND: Structural variants (SVs) represent an important source of genetic variation. One of the most critical problems in their detection is breakpoint uncertainty associated with the inability to determine their exact genomic position. Breakpoint uncertainty is a characteristic issue of structural variants detected via short-read sequencing methods and complicates subsequent population analyses. The commonly used heuristic strategy reduces this issue by clustering/merging nearby structural variants of the same type before the data from individual samples are merged. RESULTS: We compared the two most used dissimilarity measures for SV clustering in terms of Mendelian inheritance errors (MIE), kinship prediction, and deviation from Hardy-Weinberg equilibrium. We analyzed the occurrence of Mendelian-inconsistent SV clusters that can be collapsed into one Mendelian-consistent SV as a new measure of dataset consistency. We also developed a new method based on constrained clustering that explicitly identifies these types of clusters. CONCLUSIONS: We found that the dissimilarity measure based on the distance between SVs breakpoints produces slightly better results than the measure based on SVs overlap. This difference is evident in trivial and corrected clustering strategy, but not in constrained clustering strategy. However, constrained clustering strategy provided the best results in all aspects, regardless of the dissimilarity measure used.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

The vision of point-of-care PCR tests for the COVID-19 pandemic and beyond.

Infectious diseases, such as the most recent case of coronavirus disease 2019, have brought the prospect of point-of-care (POC) diagnostic tests into the spotlight. A rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. Unfortunately, despite all the advances, it still does not exist. Here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (PCR) and has come close to fulfill this vision. We summarize the requirements for the POC-PCR tests and then go on to discuss the PCR product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. We conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis.

Genome-wide miRNA profiling in plasma of pregnant women with down syndrome fetuses.

Down syndrome (DS) is one of the most common causes of intellectual disability and new approaches allowing its rapid and effective prenatal detection are being explored. In this study, we investigated the diagnostic potential of plasma microRNAs (miRNAs). This study builds upon our previous study in DS placentas, where seven miRNAs were found to be significantly up-regulated. A total of 70 first-trimester plasma samples from pregnant women were included in the present study (35 samples with DS fetuses; 35 with euploid fetuses). Genome-wide miRNA profiling was performed in the pilot study using Affymetrix GeneChip™ miRNA 4.1 Array Strips (18 samples). Selected miRNAs were then analysed in the validation study using quantitative reverse transcription PCR (RT-qPCR; 52 samples). Based on the current pilot study results (12 miRNAs), our previous research on chorionic villi samples (7 miRNAs) and the literature (4 miRNAs), a group of 23 miRNAs was selected for the validation study. Although the results of the pilot study were promising, the validation study using the more sensitive RT-qPCR technique and a larger group of samples revealed no significant differences in miRNA profiles between the compared groups. Our results suggest that testing of the first-trimester plasma miRNAs is probably not suitable for non-invasive prenatal testing (NIPT). Different results could be theoretically achieved at later gestational ages; however, such a result probably would have limited use in clinical practice.

NETosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients.

INTRODUCTION: Neutrophil extracellular traps (NETs) are formed by activated neutrophils during the process of NETosis in which the nuclear material is released into extracellular space, including DNA molecules, citrullinated histones, and neutrophil granule enzymes, such as elastase. This material forms networks that are able not only to physically entrap bacteria but also to provide elevated concentration of bactericidal components. Over the last years, it has become clear that NETs can also be formed under numerous sterile inflammatory conditions, i.e., thrombosis, cancer, SLE, atherosclerosis, and diabetes. METHOD: We reviewed studies published until July 2016 to find possible associations between elevated cell-free DNA levels in dialyzed patients and the process of NETosis and its consequences. RESULTS: The process of NETosis, its elevated activation, or impaired clearance provides the link between clinical conditions and elevated levels of cell-free DNA found in plasma after the hemodialytic procedure which itself is able to activate neutrophils via platelets and ROS formation. NETs stimulate thrombosis and endothelial damage, and their formation may contribute to the development of spectrum of comorbidities described in dialyzed patients. CONCLUSION: The study of plasma cell-free DNA levels together with markers of NETosis could contribute to the evaluation of the influence of hemodialysis on the immune system of patients.

Differentially expressed miRNAs in trisomy 21 placentas.

OBJECTIVE: Molecular pathogenesis of Down syndrome (DS) is still incompletely understood. Epigenetic mechanisms, including miRNAs gene expression regulation, belong to potential influencing factors. The aims of this study were to compare miRNAs expressions in placentas with normal and trisomic karyotype and to associate differentially expressed miRNAs with concrete biological pathways. METHODS: A total of 80 CVS samples - 41 with trisomy 21 and 39 with normal karyotype - were included in our study. Results obtained in the pilot study using real-time PCR technology and TaqMan Human miRNA Array Cards were subsequently validated on different samples using individual TaqMan miRNA Assays. RESULTS: Seven miRNAs were verified as upregulated in DS placentas (miR-99a, miR-542-5p, miR-10b, miR-125b, miR-615, let-7c and miR-654); three of these miRNAs are located on chromosome 21 (miR-99a, miR-125b and let-7c). Many essential biological processes, transcriptional regulation or apoptosis, were identified as being potentially influenced by altered miRNA levels. Moreover, miRNAs overexpressed in DS placenta apparently regulate genes involved in placenta development (GJA1, CDH11, EGF, ERVW-1, ERVFRD-1, LEP or INHA). CONCLUSION: These findings suggest the possible participation of miRNAs in Down syndrome impaired placentation and connected pregnancy pathologies. © 2016 John Wiley & Sons, Ltd.

Comparison of MicroRNA Content in Plasma and Urine Indicates the Existence of a Transrenal Passage of Selected MicroRNAs.

MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer. To examine the spectrum of miRNAs in the cell-free fraction of urine, TaqMan Human miRNA Array Card A v.2.1 was used. A set of 30 miRNAs were found that are constantly present in urine supernatants independently of sex, age and health status of the subjects. We compared this set with miRNAs found in plasma, expressed in kidney and genito-urinary tract. Our results indicate that some miRNA could be transferred from the circulation into urine.

Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer.

INTRODUCTION: Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. MATERIAL AND METHODS: In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. RESULTS: The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. CONCLUSION: Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer.

[miRNA as a new marker of diabetes mellitus and pancreatic carcinoma progression]. / miRNA jako nový ukazatel u diabetes mellitus a u rozvoje karcinomu pankreatu.

Pancreatic cancer is a disease with increasing incidence and high (and nearly unchanged) lethality that is caused mainly due to its late diagnosis. Risk factors for neoplastic transformation are especially chronic pancreatitis, diabetes mellitus, but also obesity and smoking. The search for suitable early markers becomes a key element of research in this area. Such markers could be microRNAs, short single-stranded RNA molecules functioning as regulators of translation. This article serves as a review of contemporary evidence of microRNA in diabetes mellitus and pancreatic cancer.

[Determination of body fluid based on analysis of nucleic acids]. / Urcení telních tekutin zalozené na analýze nukleových kyselin.

Recent methodological approaches of molecular genetics allow isolation of nucleic acids (DNA and RNA) from negligible forensic samples. Analysis of these molecules may be used not only for individual identification based on DNA profiling but also for the detection of origin of the body fluid which (alone or in mixture with other body fluids) forms the examined biological trace. Such an examination can contribute to the evaluation of procedural, technical and tactical value of the trace. Molecular genetic approaches discussed in the review offer new possibilities in comparison with traditional spectrum of chemical, immunological and spectroscopic tests especially with regard to the interpretation of mixtures of biological fluids and to the confirmatory character of the tests. Approaches based on reverse transcription of tissue specific mRNA and their subsequent polymerase chain reaction (PCR) and fragmentation analysis are applicable on samples containing minimal amounts of biological material. Methods for body fluid discrimination based on examination of microRNA in samples provided so far confusing results therefore further development in this field is needed. The examination of tissue specific methylation of nucleotides in selected gene sequences seems to represent a promising enrichment of the methodological spectrum. The detection of DNA sequences of tissue related bacteria has been established and it provides satisfactory results mainly in combination with above mentioned methodological approaches.

[Possibilities of molecular genetic determination of age of an individual from biological traces]. / Moznosti molekulárne genetického stanovení veku jednotlivce z biologických stop.

UNLABELLED: Application of molecular genetic methods during the examination of biological traces is irreplaceable for individual identification of their originators. However, this analysis does not provide any clues for further investigation without the possibility to compare the genetic profile of the examined trace with the profile of its potential originator. The age of a searched person represents an important entry for investigators. In this review, the recent methodical molecular genetic approaches are discussed with regards to their practical outputs leading to the estimation of biological age of an individual. The length of telomeric sequences and their attritions correlating with increasing age seemed to be very promising marker if they have been examined using Southern blot analysis. This method is not suitable for forensic casework due to the need of high amounts of DNA input. Recent methods based on quantitative polymerase chain reaction (qPCR) are applicable on samples with minimal DNA concentrations but they provide inconclusive results with regard to the age estimation based on the length of telomeres. Therefore novel methodical approaches were developed. Application of methods based on the examination of deletions in mitochondrial DNA, on the presence of transcripts of gamma hemoglobins or on the quantification of byproducts of somatic rearrangements of genes for T-cell receptors is restricted to special types of biological traces. The age dependent methylation of specific nucleotides in selected gene sequences seems to be the only promising universal marker. KEYWORDS: age estimation - molecular genetics - telomere attrition - qPCR - array technology - promoter methylation.

SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing.

This study elaborates on the design, fabrication, and data analysis details of SPEED, a recently proposed smartphone-based digital polymerase chain reaction (dPCR) device. The dPCR chips incorporate partition diameters ranging from 50 µm to 5 µm, and these partitions are organized into six distinct blocks to facilitate image processing. Due to the superior thermal conductivity of Si and its potential for mass production, the dPCR chips were fabricated on a Si substrate. A temperature control system based on a high-power density Peltier element and a preheating/cooling PCR protocol user interface shortening the thermal cycle time. The optical design employs four 470 nm light-emitting diodes as light sources, with filters and mirrors effectively managing the light emitted during PCR. An algorithm is utilized for image processing and illumination nonuniformity correction including conversion to a monochromatic format, partition identification, skew correction, and the generation of an image correction mask. We validated the device using a range of deoxyribonucleic acid targets, demonstrating its potential applicability across multiple fields. Therefore, we provide guidance and verification of the design and testing of the recently proposed SPEED device.

Application of the new insertion-deletion polymorphism kit for forensic identification and parentage testing on the Czech population.

Insertion-deletion polymorphisms (INDELs) are diallelic markers derived from a single mutation event. Their low mutation frequency makes them suitable for forensic and parentage testing. The examination of INDELs thus combines advantages of both short tandem repeats (STR) and single nucleotide polymorphisms (SNP). This type of polymorphisms may be examined using as small amplicon size as SNP (about 100 bp) but could be analyzed by techniques used for routine STR analysis. For our population study, we genotyped 55 unrelated Czech individuals. We also genotyped 11 trios to analyze DIPplex Kit (QIAGEN, Germany) suitability for parentage testing. DIPplex Kit contains 30 diallelic autosomal markers. INDELs in DIPplex Kit were tested with linkage disequilibrium test, which showed that they could be treated as independent markers. All 30 loci fulfill Hardy-Weinberg equilibrium. There were several significant differences between Czech and African populations, but no significant ones within European population. Probability of a match in the Czech population was 1 in 6.8 × 10(12); combined power of discrimination was 99.9999999999%. Average paternity index was 1.13-1.77 for each locus; combined paternity index reached about 27,000 for a set of 30 loci. We can conclude that DIPplex kit is useful as an additional panel of markers in paternity cases when mutations in STR polymorphisms are present. For application on degraded or inhibited samples, further optimization of buffer and primer concentrations is needed.

Asymptomatic abdominal aortic aneurysms show histological signs of progression: a quantitative histochemical analysis.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a serious disease due to its covert nature, relatively high prevalence and fatal prognosis in the case of rupture. To obtain new insights into AAA pathogenesis, we examined the relationships between histopathology, multiplex in vitro immunoassay data, diameter and symptomatology. METHODS: In a prospective, non-randomised study, we evaluated samples from 6 normal infrarenal aortae and 65 AAA patients (65 walls, 55 thrombi). The AAA patients were either asymptomatic (n = 44), symptomatic (n = 7) or with ruptured AAA (n = 14). The AAA diameter was classified as small (<5 cm, n = 18), medium (5-7 cm, n = 26) and large (>7 cm, n = 21). We quantified the histopathology of the AAA wall and the adjacent thrombus. We assessed the expression of proteins in the same samples. RESULTS: Asymptomatic AAAs had walls with more abundant inflammatory infiltrates, lower amounts of PAI-1, a higher number of tPA-positive elements, a tendency towards decreased collagen content, whereas the adjacent thrombi had a greater concentration of VCAM-1 and MMP-2 when compared with symptomatic AAAs. Compared with the aneurysmatic aorta, the normal aorta contained less collagen and more elastin, actin, desmin and PAI-1-positive elements; in addition, it was more vascular. Medium-sized AAAs were the most actin and vimentin rich, and large AAAs were the most vascular. CONCLUSION: Our results show that asymptomatic AAA walls often have more potentially deleterious histopathological alterations than symptomatic AAA walls. This result indicates that a progression from an asymptomatic AAA to rupture can be expected and screening patients who are at risk of rupture could be beneficial.

Methylation status of immune response genes promoters in cell-free DNA differs in hemodialyzed patients with diabetic nephropathy according to the intensity of anemia therapy.

BACKGROUND: Anemia is a major complication of end-stage renal disease. Hemodialysis itself is regarded as a stimulus activating inflammation. Pro-inflammatory cytokines are able to suppress erythropoiesis. In this pilot study, we analyzed the changes in methylation status of promoters of immune response genes in cell-free DNA to detect the differences between diabetic subjects (n = 18) with different therapeutic doses of recombinant erythropoietin. METHODS: The extent of promoter methylation of 24 genes in plasma cell-free DNA was examined before and after hemodialysis using EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity (Qiagen). RESULTS: The patients with higher methylation status of gene sequences IL13RA1, IL15, EDG3 and INHA in interdialytic interval were significantly overrepresented in the group with none or mild anemia therapy. CONCLUSION: The results are in agreement with the fact that IL13 and IL15 are known inhibitors of erythropoiesis and with considered immunomodulatory role of cell-free DNA.

Autosomal dominant polycystic kidney disease in a family with mosaicism and hypomorphic allele.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited kidney disease that results in renal failure. ADPKD is a systemic disorder with cysts and connective tissue abnormalities involving many organs. ADPKD caused by mutations in PKD1 gene is significantly more severe than the cases caused by PKD2 gene mutations. The large intra-familial variability of ADPKD highlights a role for genetic background. CASE PRESENTATION: Here we report a case of ADPKD family initially appearing unlinked to the PKD1 or PKD2 loci and the influence of mosaicism and hypomorphic allele on the variability of the clinical course of the disease. A grandmother with the PKD1 gene mutation in mosaicism (p.Val1105ArgfsX4) and with mild clinical course of ADPKD (end stage renal failure at the age of 77) seemed to have ADPKD because of PKD2 gene mutation. On the other hand, her grandson had a severe clinical course (end stage renal disease at the age of 45) in spite of the early treatment of mild hypertension. There was found by mutational analysis of PKD genes that the severe clinical course was caused by PKD1 gene frameshifting mutation inherited from his father and mildly affected grandmother in combination with inherited hypomorphic PKD1 allele with described missense mutation (p.Thr2250Met) from his clinically healthy mother. The sister with two cysts and with PKD1 hypomorphic allele became the kidney donor to her severely affected brother. CONCLUSION: We present the first case of ADPKD with the influence of mosaicism and hypomorphic allele of the PKD1 gene on clinical course of ADPKD in one family. Moreover, this report illustrates the role of molecular genetic testing in assessing young related kidney donors for patients with ADPKD.

Digital polymerase chain reaction duplexing method in a single fluorescence channel.

The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from ≈5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent channel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.

Smartphone integrated handheld (SPEED) digital polymerase chain reaction device.

We demonstrate a smartphone integrated handheld (SPEED) digital polymerase chain reaction (dPCR) device for point-of-care application. The device has dimensions of ≈100 × 200 × 35 mm3 and a weight of ≈400 g. It can perform 45 PCR cycles in ≈49 min. The device also features integrated, miniaturized modules for thermal cycling, image taking, and wireless data communication. These functions are controlled by self-developed Android-based applications. The only consumable is the developed silicon-based dPCR chip, which has the potential to be recycled. The device's precision and accuracy are comparable with commercial dPCR machines. We have verified the SPEED dPCR prototype's utility in the testing of severe acute respiratory syndrome coronavirus 2, the detection of cancer-associated gene sequences, and the confirmations of Down syndrome diagnoses. Due to its low upfront capital investment, as well as its nominal running cost, we envision that the SPEED dPCR device will help to perform cancer screenings and non-invasive prenatal tests for the general population. It will also aid in the timely identification and monitoring of infectious disease testing, thereby expediting alerts with respect to potential emerging pandemics.