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Abstract Background Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency. Results In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1. Conclusions We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.

Abstract Background Immunological and genetic findings implicate Th17 effector cytokines in the pathogenesis of inflammatory bowel disease (IBD). Expression of Th17 pathway-associated genes is mainly studied in colonic disease. The present study assessed the mRNA expression levels of Th17 effector cytokines (IL17A, IL17F, IL21, IL22 and IL26) and genes involved in differentiation (IL6, IL1B, TGFB1, IL23A and STAT3) and recruitment of Th17 cells (CCR6 and CCL20) by quantitative real-time PCR analysis of colonic and ileal biopsies from 22 healthy control subjects, 26 patients with Crohn's disease (CD) and 12 patients with ulcerative colitis (UC). Inflammation was quantified by measuring expression of the inflammatory mediators IL8 and TNF. Results Evaluation of mRNA expression levels in colonic and ileal control samples revealed that TNF, TGFB1, STAT3 and CCR6 were expressed at higher levels in the ileum than in the colon. Expression of all the Th17 pathway-associated genes was increased in inflamed colonic samples. The increased expression of these genes was predominantly observed in samples from UC patients and was associated with more intense inflammation. Although increased expression of IL17A, IL17F, IL21 and IL26 was detected in inflamed ileal samples, expression of the indispensable Th17 cell differentiation factors TGFB1 and IL23A, the signaling molecule STAT3 and the Th17 recruitment factors CCR6 and CCL20 were unchanged. Conclusions Our findings suggest that immune regulation is different in colonic and ileal disease, which might have important consequences for therapeutic intervention.

Abstract Background Cancer genomes display characteristic patterns of chromosomal imbalances, often with diagnostic and prognostic relevance. Therefore assays for genome-wide copy number screening and simultaneous detection of copy number alterations in specific chromosomal regions are of increasing importance in the diagnostic work-up of tumors. Results We tested the performance of Multiplex Amplicon Quantification, a newly developed low-cost, closed-tube and high-throughput PCR-based technique for detection of copy number alterations in regions with prognostic relevance for neuroblastoma. Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method on 52 neuroblastoma tumors showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations. Conclusion Multiplex Amplicon Quantification is a low-cost and high-throughput PCR-based technique that can reliably detect copy number alterations in regions with prognostic relevance for neuroblastoma.

Abstract Background Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. Results We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. Conclusions We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.

Abstract Background Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies. Methods We adopt and report a systematic approach to identify the most stable reference genes for miRNA expression studies by RT-qPCR in colorectal cancer (CRC). High-throughput miRNA profiling was performed on ten pairs of CRC and normal tissues. By using the mean expression value of all expressed miRNAs, we identified the most stable candidate reference genes for subsequent validation. As such the stability of a panel of miRNAs was examined on 35 tumour and 39 normal tissues. The effects of normalisers on the relative quantity of established oncogenic (miR-21 and miR-31) and tumour suppressor (miR-143 and miR-145) target miRNAs were assessed. Results In the array experiment, miR-26a, miR-345, miR-425 and miR-454 were identified as having expression profiles closest to the global mean. From a panel of six miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) and two small nucleolar RNA genes (RNU48 and Z30), miR-16 and miR-345 were identified as the most stably expressed reference genes. The combined use of miR-16 and miR-345 to normalise expression data enabled detection of a significant dysregulation of all four target miRNAs between tumour and normal colorectal tissue. Conclusions Our study demonstrates that the top six most stably expressed miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) described herein should be validated as suitable reference genes in both high-throughput and lower throughput RT-qPCR colorectal miRNA studies.

Abstract Background Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos. Findings The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated. Conclusions Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.

Abstract Background The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR. Findings The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification. Conclusion Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.

Abstract Background Recurrent loss of part of the long arm of chromosome 11 is a well established hallmark of a subtype of aggressive neuroblastomas. Despite intensive mapping efforts to localize the culprit 11q tumour suppressor gene, this search has been unsuccessful thus far as no sufficiently small critical region could be delineated for selection of candidate genes. Methods To refine the critical region of 11q loss, the chromosome 11 status of 100 primary neuroblastoma tumours and 29 cell lines was analyzed using a BAC array containing a chromosome 11 tiling path. For the genes mapping within our refined region of loss, meta-analysis on published neuroblastoma mRNA gene expression datasets was performed for candidate gene selection. The DNA methylation status of the resulting candidate gene was determined using re-expression experiments by treatment of neuroblastoma cells with the demethylating agent 5-aza-2'-deoxycytidine and bisulphite sequencing. Results Two small critical regions of loss within 11q23 at chromosomal band 11q23.1-q23.2 (1.79 Mb) and 11q23.2-q23.3 (3.72 Mb) were identified. In a first step towards further selection of candidate neuroblastoma tumour suppressor genes, we performed a meta-analysis on published expression profiles of 692 neuroblastoma tumours. Integration of the resulting candidate gene list with expression data of neuroblastoma progenitor cells pinpointed CADM1 as a compelling candidate gene. Meta-analysis indicated that CADM1 expression has prognostic significance and differential expression for the gene was noted in unfavourable neuroblastoma versus normal neuroblasts. Methylation analysis provided no evidence for a two-hit mechanism in 11q deleted cell lines. Conclusion Our study puts CADM1 forward as a strong candidate neuroblastoma suppressor gene. Further functional studies are warranted to elucidate the role of CADM1 in neuroblastoma development and to investigate the possibility of CADM1 haploinsufficiency in neuroblastoma.

Abstract Background DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers. Results To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast. Conclusion We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.

Abstract Background An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. Results The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). Conclusion This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.

Abstract Background Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11. Results In a first step, we performed high-resolution arrayCGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene. Conclusion Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.

Abstract Background The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. Results We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. Conclusion ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at http://medgen.ugent.be/arrayCGHbase/.

Abstract Background Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep. Methods A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above. Results The assay is succesfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep. Conclusions We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations.

Abstract Background Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma. Methods SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria. Results Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype. Conclusions Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis.

Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

Abstract Background Expression of human Interleukin-5 receptor alpha (hIL-5Rα) is controlled by alternative splicing, which generates two different transcripts encoding a membrane-anchored and a soluble form of the receptor, respectively. Although the study of the expression and regulation of hIL-5Rα is of crucial importance in the field of immunological processing, methods and techniques until now described lack sufficient sensitivity for detection of small differences in the expression of these isoforms. The aim of this study was to develop a reliable and sensitive real-time quantitative PCR assay to analyse the expression level of each isoform. Methods For the quantitative real-time PCR assay, two standard curves specific for each splice variant were constructed. PCR amplifications were performed on CDNA from peripheral blood, eosinophilic chronic rhinosinusitis and normal nasal tissue using a common forward and two specific reverse primers, in combination with SYBR Green I as the detection format. Results and conclusion We have developed an accurate and reliable assay for quantification of interleukin-5 receptor alpha mRNA isoforms over a broad dynamic range of input molecules. Importantly, excess of one isoform did not influence accurate quantification of the other isoform. Quantification of hIL-5Rα variants in human samples demonstrated an overexpression of both membrane-anchored and soluble encoding variants in eosinophilic chronic rhinosinusitis tissue and peripheral blood in patients with eosinophilic chronic rhinosinusitis compared to healthy subjects. The implementation of this assay will allow a better understanding of the regulatory mechanisms of the hIL-5Rα gene and hence its role in the pathogenesis of chronic inflammatory diseases.

The potential of RNA-based liquid biopsy is increasingly being recognized in diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma. This study explores the cell-free transcriptome in a humanized DLBCL patient-derived tumor xenograft (PDTX) model. Blood plasma samples (n = 171) derived from a DLBCL PDTX model, including 27 humanized (HIS) PDTX, 8 HIS non-PDTX, and 21 non-HIS PDTX non-obese diabetic (NOD)-scid IL2Rgnull (NSG) mice were collected during humanization, xenografting, treatment, and sacrifice. The mice were treated with either rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), CD20-targeted human IFNα2-based AcTaferon combined with CHOP (huCD20-Fc-AFN-CHOP), or phosphate-buffered saline (PBS). RNA was extracted using the miRNeasy serum/plasma kit and sequenced on the NovaSeq 6000 platform. RNA sequencing data of the formalin-fixed paraffin-embedded (FFPE) tissue and blood plasma samples of the original patient were included. Flow cytometry was performed on immune cells isolated from whole blood, spleen, and bone marrow. Bulk deconvolution was performed using the Tabula Sapiens v1 basis matrix. Both R-CHOP and huCD20-Fc-AFN-CHOP were able to control tumor growth in most mice. Xenograft tumor volume was strongly associated with circulating tumor RNA (ctRNA) concentration (p < 0.001, R = 0.89), as well as with the number of detected human genes (p < 0.001, R = 0.79). Abundance analysis identified tumor-specific biomarkers that were dynamically tracked during tumor growth or treatment. An 8-gene signature demonstrated high accuracy for assessing therapy response (AUC 0.92). The tumoral gene detectability in the ctRNA of the PDTX-derived plasma was associated with RNA abundance levels in the patient's tumor tissue and blood plasma (p < 0.001), confirming that tumoral gene abundance contributes to the cell-free RNA (cfRNA) profile. Decomposing the transcriptome, however, revealed high inter- and intra-mouse variability, which was lower in the HIS PDTX mice, indicating an impact of human engraftment on the stability and profile of cfRNA. Immunochemotherapy resulted in B cell depletion, and tumor clearance was reflected by a decrease in the fraction of human CD45+ cells. Lastly, bulk deconvolution provided complementary biological insights into the composition of the tumor and circulating immune system. In conclusion, the blood plasma-derived transcriptome serves as a biomarker source in a preclinical PDTX model, enables the assessment of biological pathways, and enhances the understanding of cfRNA dynamics. [ABSTRACT FROM AUTHOR]

This study comprehensively evaluated the DNA/RNA Defend Pro (DRDP) sample collection buffer, designed to inactivate and stabilize patient samples. The primary objectives were to assess DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing (RAT). Ninety-five diagnostic nasopharyngeal swab samples tested for influenza virus (influenza A), respiratory syncytial virus (RSV A), and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were 10-fold diluted with DRDP and anonymized. Initial characterization and retesting of these samples using cobas Liat confirmed 88 samples as positive, validating the presence of viral targets. Results from rapid antigen testing showed lower sensitivity compared to nucleic acid amplification testing (NAAT) but maintained perfect specificity, with 40 out of 88 positive samples by cobas Liat also testing positive for RAT. Direct RT-qPCR of DRDP-diluted samples demonstrated robust compatibility, with 72 out of 88 samples positive for cobas Liat also testing positive by direct RT-qPCR. Non-concordant results could be explained by the 200-fold lower input of extraction-free NAAT. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with extraction-free NAAT. DRDP guaranteed viral RNA stability at all temperatures for influenza A, SARS-CoV-2, and RSV A, showing stability up to 7 days at 4 °C. In conclusion, DRDP is an effective stabilizing medium compatible with direct RT-qPCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios. [ABSTRACT FROM AUTHOR]

Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew- |$t$| distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app. [ABSTRACT FROM AUTHOR]