P146 Nanopore sequencing as a maturing platform for full-length HLA genotyping

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5′ and 3′ ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoform scaffolds among ~4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5′ m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nucleotide oligomer. This oligomer adaptation method improved 5′ end sequencing and ensured correct identification of the 5′ m7G capped ends. Second, among these 5′-capped nanopore reads, we screened for ionic current signatures consistent with a 3′ polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds, most of which (257,721) aligned to protein-coding genes. Of these, 4,876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data confirmed the validity of these high-confidence RNA scaffolds.

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Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing

10.20944/preprints202108.0571.v1 ◽

2021 ◽

Author(s):

Marine Guilcher ◽

Arnaud Liehrmann ◽

Chloé Seyman ◽

Thomas Blein ◽

Guillem Rigaill ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Full Length ◽

Nanopore Sequencing ◽

Rna Seq ◽

Plastid Gene ◽

Plastid Gene Expression ◽

Short Read ◽

Transcript Processing

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short read RNA-seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is however crucial when it comes to understand the interplay between the various steps of plastid gene expression. Here, the study of the Arabidopsis leaf plastid transcriptome using Nanopore sequencing showed that many splicing and editing events were not independent but co-occurring. For a given transcript, maturation events also appeared to be chronologically ordered with splicing happening after most sites are edited.

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

10.1101/483693 ◽

2018 ◽

Cited By ~ 8

Author(s):

Adrian Viehweger ◽

Sebastian Krautwurst ◽

Kevin Lamkiewicz ◽

Ramakanth Madhugiri ◽

John Ziebuhr ◽

...

Keyword(s):

Rna Sequencing ◽

Rna Virus ◽

Consensus Sequence ◽

Full Length ◽

Viral Rna ◽

Genome Replication ◽

Nanopore Sequencing ◽

Human Coronavirus ◽

Viral Rnas ◽

Virus Genomes

Sequence analyses of RNA virus genomes remain challenging due 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 (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised 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. Using DRS, we were able to map the longest (~26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). Furthermore, 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 demonstrating 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 direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

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Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

10.1101/2021.04.30.442116 ◽

2021 ◽

Author(s):

Evangelos D. Karousis ◽

Foivos Gypas ◽

Mihaela Zavolan ◽

Oliver Muehlemann

Keyword(s):

Regulation Of Gene Expression ◽

Degradation Pathway ◽

Human Cells ◽

Full Length ◽

Main Function ◽

Nanopore Sequencing ◽

Sequencing Data ◽

Short Read ◽

Expression Levels ◽

Short Read Sequencing

Background: Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. Results: To identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD still seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. Conclusions: Long-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

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Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

10.1101/410183 ◽

2018 ◽

Cited By ~ 25

Author(s):

Alison D. Tang ◽

Cameron M. Soulette ◽

Marijke J van Baren ◽

Kevyn Hart ◽

Eva Hrabeta-Robinson ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Intron Retention ◽

Lymphocytic Leukemia ◽

Full Length ◽

Nanopore Sequencing ◽

Short Read ◽

Branch Point Sequence ◽

Full Length Transcript

AbstractSF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 have been identified with short-read RNA sequencing, a critical barrier in understanding the functional consequences of these splicing changes is that we lack the full transcript context in which these changes are occurring. Using nanopore sequencing technology, we have resequenced full-length cDNA from CLL samples with and without the hotspot SF3B1 K700E mutation, and a normal B cell. We have developed a workflow called FLAIR (Full-Length Alternative Isoform analysis of RNA), leveraging the full-length transcript sequencing data that nanopore affords. We report results from nanopore sequencing that are concordant with known SF3B1 biology from short read sequencing as well as altered intron retention events more confidently observed using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E samples identifies alternative upstream 3’ splice sites associated with SF3B1K700E. We also find downregulation of intron retention events in SF3B1K700E relative to SF3B1WT and no difference between CLL SF3B1MT and B cell, suggesting an aberrant intron retention landscape in CLL samples lacking SF3B1 mutation. With full-length isoforms, we are able to better estimate the abundance of RNA transcripts that are productive and will likely be translated versus those that are unproductive. Validation from short-read data also reveals a strong branch point sequence in these downregulated intron retention events, consistent with previously reported branch points associated with mutated SF3B1. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

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Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA binding proteins during rat heart development

10.1101/2020.07.30.229351 ◽

2020 ◽

Author(s):

Jun Cao ◽

Andrew L. Routh ◽

Muge N. Kuyumcu-Martinez

Keyword(s):

Heart Development ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Muscle Cells ◽

Full Length ◽

Actin Binding ◽

Nanopore Sequencing ◽

Mrna Isoforms

ABSTRACTAlternative splicing (AS) increases the variety of the proteome by producing multiple isoforms from a single gene. Although short-read RNA sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full length Tpm1 isoforms composed of distinct exons with specific linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected together generating predominantly muscle specific Tpm1 isoforms in adult hearts. Nanopore sequencing combined with polyA sequencing revealed that the RNA binding protein RBFOX2 controls AS of muscle specific Tpm1 isoforms and expression of TPM1 protein via terminal exon splicing impacting its polyadenylation. Furthermore, RBFOX2 regulates Tpm1 AS antagonistically to PTBP1. In sum, we defined full length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into regulation of muscle specific isoforms of Tpm1 by RNA binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle enriched genes.

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