Direct RNA nanopore sequencing of Pseudomonas aeruginosa clone C transcriptomes
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB46647 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The transcriptomes of Pseudomonas aeruginosa clone C isolates NN2 and SG17M during the mid-exponential and early stationary phases of planktonic growth were evaluated by direct RNA sequencing on the nanopore platform and compared with established short-read cDNA sequencing on the Illumina platform. Fifty to ninety percent of the sense RNAs turned out to be rRNA molecules, followed by similar proportions of mRNA transcripts and noncoding RNAs. The two platforms detected similar proportions of uncharged tRNAs and 29 yet-undescribed antisense tRNAs. For example, the rarest arginine codon was paired with the most abundant tRNAArg, and the tRNAArg gene is missing for the most frequent arginine codon. More than 90% of the antisense RNA molecules were complementary to a coding sequence. The antisense RNAs were evenly distributed in the genomes. Direct RNA sequencing identified more than 4,000 distinct nonoverlapping antisense RNAs during exponential and stationary growth. Besides highly expressed small antisense RNAs less than 200 bases in size, a population of longer antisense RNAs was sequenced that covered a broad range (a few hundred to thousands of bases) and could be complementary to a contig of several genes. In summary, direct RNA sequencing identified yet-undescribed RNA molecules and an unexpected composition of the pools of tRNAs and sense and antisense RNAs. IMPORTANCE Genome-wide gene expression of bacteria is commonly studied by high-throughput sequencing of size-selected cDNA fragment libraries of reverse-transcribed RNA preparations. However, the depletion of rRNAs, enzymatic reverse transcription, and the fragmentation, size selection, and amplification during library preparation lead to inevitable losses of information about the initial composition of the RNA pool. We demonstrate that direct RNA sequencing on the Nanopore platform can overcome these limitations. Nanopore sequencing of total RNA yielded novel insights into the Pseudomonas aeruginosa transcriptome that-if replicated in other species-will change our view of the bacterial RNA world. The discovery of sense-antisense pairs of transfer-messenger RNA (tmRNA), tRNAs, and mRNAs indicates a further and unknown level of gene regulation in bacteria.
| S-EPMC8765398 | biostudies-literature
Project description:The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyze the 5' and 3' ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N6 -methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as a template, we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Productive infection of primary fibroblasts with herpes simplex virus Support Protocol: Cell passage and plating of primary fibroblasts Basic Protocol 2: Preparation and sequencing of dRNA-seq libraries from virus-infected cells Basic Protocol 3: Processing, alignment, and analysis of dRNA-seq datasets.
| S-EPMC7187905 | biostudies-literature
Project description:Detection of circRNA through Nanopore direct RNA sequencing
| PRJEB52141 | ENA
Project description:Long-read nanopore sequencing has emerged as a potent tool for studying RNA modifications. However, the detection of N4-acetylcytidine (ac4C) based on nanopore sequencing remains largely unexplored. Here, we introduce ac4Cnet, a deep learning frame utilizing Oxford Nanopore direct RNA sequencing to accurately identify ac4C sites. Our methodology involves training ac4Cnet capable of distinguishing ac4C from unmodified cytidine and 5-methylcytosine (m5C), as well as estimating the modification rate at each ac4C site. We demonstrate the robustness of our approach through validations on independent in vitro datasets and a human cell line, highlighting its versatility and potential for advancing the study of ac4C modifications.
2025-02-08 | GSE267558 | GEO
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.
2023-03-01 | GSE195618 | GEO
Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.
2019-03-14 | E-MTAB-7778 | biostudies-arrayexpress
Project description:To detect the modifed bases in SINEUP RNA, we compared chemically modified in vitro transcribed (IVT) SINEUP-GFP RNA and in-cell transcribed (ICT) SINEUP RNA from SINEUP-GFP and sense EGFP co-transfected HEK293T/17 cells. Comparative study of Nanopore direct RNA sequencing data from non-modified and modified IVT samples against the data from ICT SINEUP RNA sample revealed modified k-mers positions in SINEUP RNA in the cell.
2023-02-04 | GSE224018 | GEO
Project description:Anonynous donor kidneys were sequenced using the Nanopore long-read direct cDNA sequencing kit (SQK-DCS109) and R9.4.1 flow cells.
2024-11-10 | GSE281264 | GEO
Project description:RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.
| S-EPMC8664944 | biostudies-literature
Project description:RNA modifications are a common occurrence across all domains of life. Several chemical modifications, including N6-methyladenosine, have also been found in viral transcripts and viral RNA genomes. Some of the modifications increase the viral replication efficiency while also helping the virus to evade the host immune system. Nonetheless, there are numerous examples in which the host's RNA modification enzymes function as antiviral factors. Although established methods like MeRIP-seq and miCLIP can provide a transcriptome- wide overview of how viral RNA is modified, it is difficult to distinguish between the complex overlapping viral transcript isoforms using the short read-based techniques. Nanopore direct RNA sequencing (DRS) provides both long reads and direct signal readings, which may carry information about the modifications. Here, we describe a refined protocol for analyzing the RNA modifications in viral transcriptomes using nanopore technology.
| S-EPMC9400574 | biostudies-literature