Project description:Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.
Project description:State-of-the-art algorithms for m6A detection and quantification via nanopore direct RNA sequencing have been continuously developed, little is known about their capacities and limitations, which makes a comprehensive assessment in urgent need. Therefore, we performed comprehensive benchmarking of 10 computational tools relying on current-based and base-calling “errors” strategies for m6A detection by nanopore sequencing.
Project description:This series includes 278 microarrays used to detect respiratory viruses in a set of nasopharyngeal lavage specimens from children with respiratory tract infections Objective: To assess the utility of a pan-viral DNA microarray platform (Virochip) in the detection of viruses associated with pediatric respiratory tract infections. Study Design: The Virochip was compared to conventional clinical direct fluorescent antibody (DFA) and PCR-based testing for the detection of respiratory viruses in 278 consecutive nasopharyngeal aspirate samples from 222 children. Results: The Virochip was superior in performance to DFA, showing a 19% increase in the detection of 7 respiratory viruses included in standard DFA panels, and was similar to virus-specific PCR (sensitivity 85-90%, specificity 99%, PPV 94-96%, NPV 97-98%) in the detection of respiratory syncytial virus, influenza A, and rhino-/enteroviruses. The Virochip also detected viruses not routinely tested for or missed by DFA and PCR, as well as double infections and infections in critically ill patients that DFA failed to detect. Conclusions: Given its favorable sensitivity and specificity profile and greatly expanded spectrum of detection, microarray-based viral testing holds promise for clinical diagnosis of pediatric respiratory tract infections. Keywords: viral detection
Project description:This series includes 278 microarrays used to detect respiratory viruses in a set of nasopharyngeal lavage specimens from children with respiratory tract infections Objective: To assess the utility of a pan-viral DNA microarray platform (Virochip) in the detection of viruses associated with pediatric respiratory tract infections. Study Design: The Virochip was compared to conventional clinical direct fluorescent antibody (DFA) and PCR-based testing for the detection of respiratory viruses in 278 consecutive nasopharyngeal aspirate samples from 222 children. Results: The Virochip was superior in performance to DFA, showing a 19% increase in the detection of 7 respiratory viruses included in standard DFA panels, and was similar to virus-specific PCR (sensitivity 85-90%, specificity 99%, PPV 94-96%, NPV 97-98%) in the detection of respiratory syncytial virus, influenza A, and rhino-/enteroviruses. The Virochip also detected viruses not routinely tested for or missed by DFA and PCR, as well as double infections and infections in critically ill patients that DFA failed to detect. Conclusions: Given its favorable sensitivity and specificity profile and greatly expanded spectrum of detection, microarray-based viral testing holds promise for clinical diagnosis of pediatric respiratory tract infections. Keywords: viral detection The series includes 278 clinical specimens
Project description:To evaluate targeted MinION next generation sequencing as a diagnostic method for detection of pathogens in human blood and plasma, human blood or plasma samples were spiked with measured amounts of viruses, bacteria, protozoan parasites or tested pathogen-free as negative controls. Nucleic acid was extracted from samples and PCR amplification performed in multiplex primer pools with a procedure described in ArrayExpress experiment submission ID 18379. The PCR products were used for library preparation. The libraries sequenced on an Oxford Nanopore MinION. The passed reads aligned with a custom reference file to determine the identity of the pathogen in the sample.
Project description:Chinese yam (Dioscorea opposita Thumb.) is a highly nutritional perennial herbaceous rhizome. N-glycoforms from Chinese yam glycoprotein were analyzed, and the major glycoprotein in yam be identificated.
2019-10-28 | PXD015888 | Pride
Project description:Nanopore adaptive sampling for detection of tick-borne RNA viruses
Project description:We designed a pan-Microbial Detection Array (MDA) to detect all known viruses (including phage), bacteria, and plasmids. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms. The array has wider coverage of bacterial and viral targets based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. In blinded lab testing on spiked samples with single or multiple viruses, the MDA was able to correctly identify species or strains. In clinical fecal, serum, and respiratory samples, the MDA was able to detect and characterize multiple viruses, phage, and bacteria in a sample to the family and species level, as confirmed by PCR. Testing of microbial detection array with mixtures of known viruses, blinded clinical samples and viral cell culture samples.
Project description:We designed a pan-Microbial Detection Array (MDA) to detect all known viruses (including phage), bacteria, and plasmids. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms. The array has wider coverage of bacterial and viral targets based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. In blinded lab testing on spiked samples with single or multiple viruses, the MDA was able to correctly identify species or strains. In clinical fecal, serum, and respiratory samples, the MDA was able to detect and characterize multiple viruses, phage, and bacteria in a sample to the family and species level, as confirmed by PCR. Testing of microbial detection array with mixtures of known viruses, blinded clinical samples and viral cell culture samples.
Project description:Using Oxford Nanopore Sequencing, we sequenced the methylome of wild type zebrafish (TL background) forebrain. Forebrains from six individuals were profiled, enabling direct detection of multiple DNA base modifications at single-base resolution. The resulting dataset includes CpG-associated 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), as well as non-CpG 5mC and N6-methyladenine (6mA). NOTE: Raw Oxford Nanopore sequencing reads have been deposited in the European Nucleotide Archive under accession PRJEB108899 - this record was created independently by the submitter, in case of access issues, please contact the submitter directly.