MethPanel: a parallel pipeline and interactive analysis tool for multiplex bisulphite PCR sequencing to assess DNA methylation biomarker panels for disease detection.
Ontology highlight
ABSTRACT: Summary
DNA methylation patterns in a cell are associated with gene expression and the phenotype of a cell, including disease states. Bisulphite PCR sequencing is commonly used to assess the methylation profile of genomic regions between different cells. Here we have developed MethPanel, a computational pipeline with an interactive graphical interface to rapidly analyse multiplex bisulphite PCR sequencing data. MethPanel comprises a complete analysis workflow from genomic alignment to DNA methylation calling and supports an unlimited number of PCR amplicons and input samples. MethPanel offers important and unique features, such as calculation of a epipolymorphism score and bisulphite PCR bias correction capabilities, and is designed so that the methylation data from all samples can be processed in parallel. The outputs are automatically forwarded to a shinyApp for convenient display, visualisation and remotely sharing data with collaborators and clinicians.Availability
MethPanel is freely available at https://github.com/thinhong/MethPanel.Supplementary information
Supplementary data are available at Bioinformatics online.
SUBMITTER: Luu PL
PROVIDER: S-EPMC8352503 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundDNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material.ResultsHere, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1-5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA.ConclusionsThe MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.
| S-EPMC7310104 | biostudies-literature
Project description:UNLABELLED: PREMISE OF THE STUDY:New microsatellites were developed for the seagrass Thalassia hemprichii (Hydrocharitaceae), a long-lived seagrass species that is found throughout the shallow waters of tropical and subtropical Indo-West Pacific. Three multiplex PCR panels were designed utilizing new and previously developed markers, resulting in a toolkit for generating a 16-locus genotype. • METHODS AND RESULTS:Through the use of microsatellite enrichment and next-generation sequencing, 16 new, validated, polymorphic microsatellite markers were isolated. Diversity was between two and four alleles per locus totaling 36 alleles. These markers, plus previously developed microsatellite markers for T. hemprichii and T. testudinum, were tested for suitability in multiplex PCR panels. • CONCLUSIONS:The generation of an easily replicated suite of multiplex panels of codominant molecular markers will allow for high-resolution and detailed genetic structure analysis and clonality assessment with minimal genotyping costs. We suggest the establishment of a T. hemprichii primer convention for the unification of future data sets.
| S-EPMC4222546 | biostudies-literature
Project description:Next-generation sequencing has greatly promoted the investigation of single nucleotide polymorphisms, while studies of simple sequence repeats are sharply decreasing. However, simple sequence repeats still present some advantages in conservation genetics. In this study, an end-to-end pipeline referred to as MultiplexSSR was established to develop multiplex PCR assays in batches with highly polymorphic simple sequence repeats for capillary platforms from resequencing data. The distribution of single sequence repeats in the genome, the error profiles of genotypes and allelotypes, and the increase in the allele length range depending on the number of individuals were investigated. A total of 98% of single sequence repeats presented lengths of less than 100 bp. The error rate of the genotyping and allelotyping of dimeric patterns was ten times higher than those for other patterns. The error rate of allelotyping was less than that of genotyping. The allele length range reached approximate saturation with 10 individuals. This pipeline uses allele numbers to select highly polymorphic loci, masks loci with variation, and applies in silico PCR to improve primer specificity. The application of the developed multiplex SSR-PCR assays validated the pipeline's robustness, showing higher polymorphism and stability for the developed simple sequence repeats and a lower cost for genotyping and providing low-depth resequencing data from less than a dozen individuals for the development of markers. This pipeline fills the gap between next-generation sequencing and multiplex SSR-PCR.
| S-EPMC7083706 | biostudies-literature
Project description:Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.
| S-EPMC5419558 | biostudies-literature
Project description:ObjectiveTo design and evaluate an interactive data quality (DQ) characterization tool focused on fitness-for-use completeness measures to support researchers' assessment of a dataset.Materials and methodsDesign requirements were identified through a conceptual framework on DQ, literature review, and interviews. The prototype of the tool was developed based on the requirements gathered and was further refined by domain experts. The Fitness-for-Use Tool was evaluated through a within-subjects controlled experiment comparing it with a baseline tool that provides information on missing data based on intrinsic DQ measures. The tools were evaluated on task performance and perceived usability.ResultsThe Fitness-for-Use Tool allows users to define data completeness by customizing the measures and its thresholds to fit their research task and provides a data summary based on the customized definition. Using the Fitness-for-Use Tool, study participants were able to accurately complete fitness-for-use assessment in less time than when using the Intrinsic DQ Tool. The study participants perceived that the Fitness-for-Use Tool was more useful in determining the fitness-for-use of a dataset than the Intrinsic DQ Tool.DiscussionIncorporating fitness-for-use measures in a DQ characterization tool could provide data summary that meets researchers needs. The design features identified in this study has potential to be applied to other biomedical data types.ConclusionA tool that summarizes a dataset in terms of fitness-for-use dimensions and measures specific to a research question supports dataset assessment better than a tool that only presents information on intrinsic DQ measures.
| S-EPMC9667174 | biostudies-literature
Project description:Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the "current standard" of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.
| S-EPMC5648699 | biostudies-other
Project description:BACKGROUND:Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%-5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce noise via deep sequencing unavoidably reduce throughput and increase cost. Here we provide proof of principle that coamplification at lower denaturation temperature (COLD)-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS:We have developed a multiplex temperature-tolerant COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. After multiplex preamplification from genomic DNA, we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for melting temperature (Tm)-increasing mutations over all amplicons, in a single tube. Therefore, in separate reactions, both Tm-decreasing and Tm-increasing mutations are enriched. RESULTS:Using custom-made and commercial gene panels containing 8, 50, 190, or 16 000 amplicons, we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of deoxyinosine triphosphate (dITP)/2,6-diaminopurine triphosphate (dDTP) in place of deoxyguanosine triphosphate (dGTP)/deoxyadenosine triphosphate (dATP) enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit of detection of approximately 0.01%-0.1% mutation abundance by use of Ion-Torrent and 0.1%-0.3% by use of Sanger sequencing. CONCLUSIONS:Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single-tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.
| S-EPMC4281501 | biostudies-literature
Project description:The development of automated servers to predict the three-dimensional structure of proteins has seen much progress over the years. These servers make calculations simpler, but largely exclude users from the process. In this study, we present the PRotein Interactive MOdeling (PRIMO) pipeline for homology modeling of protein monomers. The pipeline eases the multi-step modeling process, and reduces the workload required by the user, while still allowing engagement from the user during every step. Default parameters are given for each step, which can either be modified or supplemented with additional external input. PRIMO has been designed for users of varying levels of experience with homology modeling. The pipeline incorporates a user-friendly interface that makes it easy to alter parameters used during modeling. During each stage of the modeling process, the site provides suggestions for novice users to improve the quality of their models. PRIMO provides functionality that allows users to also model ligands and ions in complex with their protein targets. Herein, we assess the accuracy of the fully automated capabilities of the server, including a comparative analysis of the available alignment programs, as well as of the refinement levels used during modeling. The tests presented here demonstrate the reliability of the PRIMO server when producing a large number of protein models. While PRIMO does focus on user involvement in the homology modeling process, the results indicate that in the presence of suitable templates, good quality models can be produced even without user intervention. This gives an idea of the base level accuracy of PRIMO, which users can improve upon by adjusting parameters in their modeling runs. The accuracy of PRIMO's automated scripts is being continuously evaluated by the CAMEO (Continuous Automated Model EvaluatiOn) project. The PRIMO site is free for non-commercial use and can be accessed at https://primo.rubi.ru.ac.za/.
| S-EPMC5113968 | biostudies-literature
Project description:In forensic casework, compromised samples often possess limited or degraded nuclear DNA, rendering mitochondrial DNA a more feasible option for forensic DNA analyses. The emergence of massively parallel sequencing (MPS) has enabled the recovery of extensive sequence information from very low quantities of DNA. We have developed a multiplex PCR method that amplifies the complete mitochondrial genome in a range of forensically relevant samples including single cells, cremated remains, bone, maggot and hairs isolated from dust bunnies. Following library preparation, MPS yields complete or nearly complete mitochondrial genome coverage for all samples. To confirm concordance between sample types and between sequencing platforms, we compared sequencing results from hair and buccal swabs from two references. Low initial DNA input into the multiplex PCR allows for conservation of precious DNA while MPS maximizes recovery of genetic information.
| S-EPMC6222269 | biostudies-literature
Project description:Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay was developed which combines different types of molecular markers in a high-throughput microsphere suspension array. The 52 molecular markers include prophage genes, amplified fragment length polymorphism (AFLP) elements, Salmonella genomic island 1 (SGI1), allantoinase gene allB, MLVA locus STTR10, antibiotic resistance genes, single nucleotide polymorphisms (SNPs) and phase 2 flagellar gene fljB. The in vitro stability of these markers was confirmed in a serial passage experiment. The validation of the MOL-PCR assay for subtyping of S. Typhimurium and S. 1,4,[5],12:i:- on 519 isolates shows that the method is rapid, reproducible, flexible, accessible, easy to use and relatively inexpensive. Additionally, a 100 % typeability and a discriminatory power equivalent to that of phage typing were observed, and epidemiological concordance was assessed on isolates of 2 different outbreaks. Furthermore, a data analysis method is provided so that the MOL-PCR assay allows for objective, computerised data analysis and data interpretation of which the results can be easily exchanged between different laboratories in an international surveillance network.
| S-EPMC4561068 | biostudies-literature