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At least 19 recordsLinked to original sources
PubMed2026

New insights on Plasmodium gene expression from direct RNA sequencing.

Oxford Nanopore Technology (ONT) direct RNA sequencing enables the sequencing of native RNA molecules without cDNA conversion. The long-read approach captures full-length reads spanning entire genes and has transformed the study of gene expression in Plasmodium parasites by enabling analysis of untranslated regions, isoforms, and alternative splicing. In addition, ONT provides unique insights into non-coding RNAs, RNA modifications, and polyadenylated tail dynamics, which are expanding our understanding of post-transcriptional regulation in Plasmodium, including processes beyond translational repression in gametocytes and sporozoites. Here, we discuss the past and future applications of direct RNA sequencing in Plasmodium research and highlight its advantages, limitations, and future prospects.

Oxford Nanopore Technology↗
PubMed2026

ModiCal: A Targeted Calibration Workflow for Site-Specific m5C Validation by Nanopore Direct RNA Sequencing.

Accurate identification of RNA 5-methylcytidine (m5C) at the single-nucleotide resolution remains a central challenge in nanopore direct RNA sequencing (DRS). Current global scanning and modification-aware basecalling methods enable transcriptome-wide profiling but often yield high false-positive rates and lack site-specific accuracy. To address this, we repurposed ModiDeC, originally a de novo multimodification classifier, into a targeted, high-precision validation tool for RNA modification sites with prior biochemical knowledge. This was implemented through a three-step calibration workflow that alternates between biochemical and computational modules using the well-characterized m5C2278 site in 25S rRNA as a starting point. Baseline training uses short synthetic RNAs carrying either a methylated or unmodified C2278 as ground truth, followed by IVT-derived calibration and validation in methyltransferase knockout yeast. The baseline model accurately detected the bona fide m5C2278 site but initially produced off-target predictions. Iterative retraining with unmodified IVT signals progressively reduced and ultimately eliminated false positives while maintaining a strong signal at the bona fide site. The final model retained enzyme-dependent detection in wild-type versus knockout yeast and, when explicitly targeted, was also able to detect the second rRNA site, C2870, which remained invisible in the initial analysis. Application to native human prerRNA processing intermediates further resolved two distinct m5C deposition regimes on 28S rRNA, while generalization to dengue virus genomic RNA confirmed that the same calibration logic transfers across diverse RNA contexts. Together, this study establishes a reproducible and transferable framework that integrates biochemical validation with iterative neural network refinement, providing a route toward reliable site-specific m5C confirmation by nanopore direct RNA sequencing.

RNA Methylation↗
PubMed1980

An improved direct RNA sequence method; its application to Vicia faba 5.8S ribosomal RNA.

We have developed a direct read-off sequencing procedure, based on the method of Stanley and Vassilenko using E. coli 5S ribosomal RNA as a model compound. Radioactive bands were transferred from an acrylamide gel fractionation in the first dimension onto a DEAE-cellulose thin layer plate. After in situ enzymatic digestion with RNase T2, mononucleoside 3',5'-diphosphates were separated in the second dimension by electrophoresis at pH 2.3. Using this two-dimensional procedure the entire sequence of 163 residues of the previously unknown Vicia faba (broad bean) 5.8S ribosomal RNA was deduced.

Base Sequence↗
PubMed2026

DirectASRM: uncovering allele-specific post-transcriptional RNA modifications through direct RNA sequencing.

SUMMARY: We developed DirectASRM, a comprehensive database for the systematic identification, integration, and annotation of allele-specific RNA modifications (ASRMs) from direct RNA sequencing data. DirectASRM enables single-base, transcript-level detection of ASRMs across multiple RNA modification types, diverse organisms and condition-specific contexts. The database further evaluates the confidence of each ASRM-SNP pair association within isoform context by jointly considering statistical evidence of allelic modification imbalance and independent support from external next-generation sequencing (NGS) - based RNA modification resources. DirectASRM also provides extensive functional annotations for ASRMs and their associated variants, including intra-sample transcript-level allele-specific expression (ASE) and allele-specific splicing, as well as additional post-transcriptional regulatory features such as miRNA binding, circRNA, RNA-protein interactions, and disease relevance. Overall, DirectASRM serves as a comprehensive resource that supports systematic investigation of the potential functional impact of genetic variants in epitranscriptomic regulation. AVAILABILITY AND IMPLEMENTATION: DirectASRM database is freely accessible at http://modinfor.com/DirectASRM/. DirectASRM pipeline is available at GitHub (https://github.com/jiayin1101/DirectASRM_pipeline) and Zenodo (DOI: https://doi.org/10.5281/zenodo.19876077).

Alleles↗
PubMed1994

Intron sequence directs RNA editing of the glutamate receptor subunit GluR2 coding sequence.

The Ca2+ permeability and the rectifying properties of the glutamate receptors assembled from the subunits GluR1-GluR4 depend upon a critical Arg in the GluR2 subunit located in a domain that has been proposed to span the membrane. The GluR2 subunit gene encodes a Gln (CAG) at this position, whereas the mRNA is edited so that it encodes an Arg (CGG) at this position [Sommer, B., Kohler, M., Sprengel, R. & Seeburg, P. H. (1991) Cell 67, 11-20]. The editing process is specific since only the GluR2 subunit RNA is edited even though the GluR1, GluR3, and GluR4 RNAs have a similar sequence. We show that this selective RNA editing depends upon a critical intron sequence in the GluR2 gene. This critical intron sequence is sufficient to cause editing of the GluR3 subunit exon in a chimera minigene constructed so that the GluR3 exon is placed upstream to the GluR2 intron sequence. Transfections of a neuronal cell line, N2a, with minigene constructs encoding different fragments of the GluR2 gene demonstrate that the 5' part of the 3' intron is essential for editing. Part of the exon and this critical intron sequence contains an inverted repeat that can fold into a structure consisting of three helical elements. Similar conclusions were reached by Higuchi, M., Single, F. n., Köhler, M., Sommer, B., Sprengel, R. & Seeburg, P. H. [(1993) Cell 75, 1361-1370]. These experiments demonstrate that the low Ca2+ permeability of the ionotropic non-N-methyl-D-aspartate glutamate receptors depends upon RNA editing, which requires a sequence in an intron 3' to the exon.

Algorithms↗
PubMed2026

Characterization of METTL3/14-mediated m6A modification in human transcriptome using Nanopore direct RNA sequencing.

Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m6A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m6A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3/METTL14 to cellular functions and disease.

Humans↗
PubMed1984

Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis.

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.

Animals↗
PubMed1990

Molecular cloning of the mammalian fatty acid synthase gene and identification of the promoter region.

Rat genomic clones encompassing the entire fatty acid synthase gene have been isolated and characterized. The gene is present in a single copy of approx. 20 kb. Genomic DNA sequencing, direct RNA sequencing and S1 nuclease analysis showed that transcription is initiated primarily 1274 nucleotides upstream from the translation start site and that the 87-nucleotide-long 5'-untranslated mRNA sequence is the same in liver, lung and mammary gland. The 5'-flanking region and first intron contain several sequence elements which may be involved in the transcriptional regulation of this gene.

Animals↗
PubMed2004

Evaluation of the phylogenetic position of the planctomycete 'Rhodopirellula baltica' SH 1 by means of concatenated ribosomal protein sequences, DNA-directed RNA polymerase subunit sequences and whole genome trees.

In recent years, the planctomycetes have been recognized as a phylum of environmentally important bacteria with habitats ranging from soil and freshwater to marine ecosystems. The planctomycetes form an independent phylum within the bacterial domain, whose exact phylogenetic position remains controversial. With the completion of sequencing of the genome of 'Rhodopirellula baltica' SH 1, it is now possible to re-evaluate the phylogeny of the planctomycetes based on multiple genes and genome trees in addition to single genes like the 16S rRNA or the elongation factor Tu. Here, evidence is presented based on the concatenated amino acid sequences of ribosomal proteins and DNA-directed RNA polymerase subunits from 'Rhodopirellula baltica' SH 1 and more than 90 other publicly available genomes that support a relationship of the Planctomycetes and the Chlamydiae. Affiliation of 'Rhodopirellula baltica' SH 1 and the Chlamydiae was reasonably stable regarding site selection since, during stepwise filtering of less-conserved sites from the alignments, it was only broken when rigorous filtering was applied. In a few cases, 'Rhodopirellula baltica' SH 1 shifted to a deep branching position adjacent to the Thermotoga/Aquifex clade. These findings are in agreement with recent publications, but the deep branching position was dependent on site selection and treeing algorithm and thus not stable. A genome tree calculated from normalized BLASTP scores did not confirm a close relationship of 'Rhodopirellula baltica' SH 1 and the Chlamydiae, but also indicated that the Planctomycetes do not emerge at the very root of the Bacteria. Therefore, these analyses rather contradict a deep branching position of the Planctomycetes within the bacterial domain and reaffirm their earlier proposed relatedness to the Chlamydiae.

Bacteria↗
PubMed2007

Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases.

PURPOSE: Long QT Syndrome, Marfan Syndrome, hypertrophic and dilated cardiomyopathy are caused by mutations in large, multi-exon genes that are principally expressed in cardiovascular tissues. Genetic testing for these disorders is labor-intensive and expensive. We sought to develop a more rapid, comprehensive, and cost-effective approach. METHODS: Paired whole blood samples were collected into tubes with or without an RNA-preserving solution, and harvested for whole blood RNA or leukocyte DNA, respectively. Large overlapping cDNA fragments from KCNQ1 and KCNH2 (Long QT Syndrome), MYBPC3 (hypertrophic and dilated cardiomyopathy), or FBN1 (Marfan Syndrome) were amplified from RNA and directly sequenced. Variants were confirmed in leukocyte DNA. RESULTS: All 4 transcripts were amplified and sequenced from whole blood mRNA. Six known and 2 novel mutations were first identified from RNA of 10 probands, and later confirmed in genomic DNA, at considerable savings in time and cost. In one patient with MFS, RNA sequencing directly identified a splicing mutation. Results from RNA and DNA were concordant for single nucleotide polymorphisms at the same loci. CONCLUSION: Taking advantage of new whole blood RNA stabilization methods, we have designed a cost-effective, comprehensive method for mutation detection that should significantly facilitate clinical genetic testing in four lethal cardiovascular disorders.

Adolescent↗
PubMed1983

High spontaneous mutation rate of Rous sarcoma virus demonstrated by direct sequencing of the RNA genome.

Direct and extensive sequencing of RSV RNA genome is reported. More than 10,000 nt of the T1 RNase resistant RSV RNA fragments (1) have been sequenced and shown to cover 3900 nt of RSV genome. The frequent sequence variations found indicate that RSV supports a very high incidence of spontaneous mutations in the course of replication, one very probable cause of the genetic diversity among the avian retroviruses. Sequences of the structured RSV RNAs allowed us also to precisely characterize the structured domains of the retroviral genome and show that the src gene is not structured.

Amino Acid Sequence↗
PubMed1992

[Importance of PCR in the diagnosis of hepatitis C].

The identification of hepatitis C virus, based on DNA amplification, gives a precise estimation of the prevalence of the most frequent agent of NANB hepatitis. The first ELISA allowing the detection of anti-HCV antibodies, had too many false positive results and required the development of more sensitive and specific assays to confirm its results. PCR, allowing the hepatitis C virus diagnosis by showing directly HCV RNA sequences, offers a complementary approach to immunoserological tests. In blood donors with anti-HCV antibodies and with indeterminate or negative confirmatory tests, the finding of HCV RNA sequences reveals serum infectivity. During acute hepatitis, the delay in the appearance of anti HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on-going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness and a third one distinct from the previous two. However, its limits and constraints imply that PCR must not be considered as a routine assay. This emphasizes the need for more simple and rapid diagnostic tests, allowing the detection of HCV antigens and, as in hepatitis B, the progressive unravelling of the life cycle of HCV.

Animals↗
PubMed1989

Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA.

Ribosomal RNA sequences are an appealing target for bacterial classification as well as for development of group- or species-specific DNA probes. Using the polymerase chain reaction and synthetic primers, the feasibility of this gene amplification technique for rapid sequence determination of the major 16S ribosomal RNA domains from small amounts of input DNA is demonstrated. Information useful for phylogenetic classification as well as for construction of specific DNA probes may be obtained by comparison with known sequences.

Bacteria↗
PubMed2019

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Cell Line↗
PubMed1980

Phy M: an RNase activity specific for U and A residues useful in RNA sequence analysis.

Physarum polycephalum, an acellular slime mold, produces an endoribonuclease activity (Phy M) useful in direct RNA sequence determination. Under conditions previously described for direct enzymatic RNA sequencing (7M urea, 50 degrees C, pH 5.0)1 Phy M cleaves almost exclusively and uniformly at Up-N and Ap-N. In the absence of urea Up-N, Ap-N, and Gp-N are attacked in a sequence specific way identical to that found with RNase Phy I2 digests performed under the same reaction conditions.

Adenine↗
PubMed1996

Cucumber mosaic virus RNA 5 is a mixed population derived from the conserved 3'-terminal regions of genomic RNAs 2 and 3.

Analysis of encapsidated RNA from the Q strain of CMV (Q-CMV) has indicated the presence of a discreet population of molecules of approximately 300 nt termed RNA 5 (Peden, K.W.C., and Symons, R.H., Virology 53,487-492, 1973.). Q-CMV RNA 5 was isolated and the 5'-end sequence was determined by direct RNA sequencing. This sequence corresponded to the exact beginning of the imperfectly conserved 3'-terminal region of genomic RNAs 1, 2, and 3. A probe generated from RNA 3 consisting of the last 130 nt of this region hybridised to RNA 5. Oligonucleotides containing sequences from the 5'- and 3'-ends of the conserved region were used to generate RNA 5 cDNA clones by RT-PCR on gel-purified RNA 5. Sequencing of these clones and primer extension analysis of transcripts (derived from the cDNA clones) and purified RNA 5 indicated that RNA 5 consists of the conserved 3'-terminal region of genomic RNAs 2 and 3.

Base Sequence↗
PubMed2024

Nanopore sequencing to detect A-to-I editing sites.

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.

Inosine↗