Advances in nanopore direct RNA sequencing

Jain, Miten; Abu-Shumays, Robin; Olsen, Hugh E.; Akeson, Mark

doi:10.1038/s41592-022-01633-w

Cited by 79 publications

(90 citation statements)

References 37 publications

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“…These developments enable us to confidently identify four previously unreported mRNA modifications in S. cerevisiae (m 1 G, m 2 G, m 2 2 G and m 5 U), demonstrating the utility of applying LC-MS/MS to discover and quantify mRNA modifications. Going forward, integrating quantitative LC-MS/MS approaches, like the one presented here, with sequencing based methodologies for transcriptome-wide modification mapping can facilitate the development of rigorous platforms to studying mRNA modifications 69,[109][110][111][112][113][114][115][116][117][118] . In addition to revealing the enzymes that incorporate these modifications, we also demonstrate that the presence of m 1 G, m 2 G, and m 5 U in mRNA can alter translation.…”

Section: Discussionmentioning

confidence: 99%

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

et al. 2023

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Section: Discussionmentioning

confidence: 99%

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

et al. 2023

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“…Since DRS starts from the 3’-end of poly(A)-tailed RNAs, this results in a 3’-end bias. Furthermore, DRS also suffers from a 5’-end ambiguity because the sequenced RNA strands are released from the nanopore about 10–12nt before they reach the 5’-terminus (Jain et al, 2022). Artificial 5’- and 3’-termini are randomly distributed and generally represented by only single reads.…”

Section: Resultsmentioning

confidence: 99%

“…Together, the data demonstrate that DRS can provide a more comprehensive and bias-free understanding of the T. brucei transcriptome. Provided that the current basecall accuracy and genome coverage limitations can be overcome (Jain et al, 2022), it should be possible to gain further insight into the plasticity of RNA processing reactions in T. brucei , which should accelerate our understanding of the RNA biology of the parasite.…”

Section: Discussionmentioning

confidence: 99%

Nanopore-based direct RNA sequencing of theTrypanosoma bruceitranscriptome identifies novel lncRNAs

Kruse

Göringer

2023

Preprint

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Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression posttranscriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example, by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized today. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known because of the inherent cDNA recoding and DNA amplification limitations of short-read RNA sequencing. Here we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome T. brucei with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 109 of which are stage-specifically expressed. We also present insights into the complexity of the T. brucei transcriptome, including alternative transcriptional start and stop sites and potential transcript isoforms to provide a bias-free understanding of the intricate RNA landscape in T. brucei.

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“…Since DRS starts from the 3′-end of poly(A)-tailed RNAs, this results in a 3′-end bias. Furthermore, DRS also suffers from a 5′-end ambiguity, because the sequenced RNA strands are released from the nanopore about 10–12 nt before they reach the 5′-terminus [ 40 ]. Artificial 5′- and 3′-termini are randomly distributed and generally represented by only single reads.…”

Section: Resultsmentioning

confidence: 99%

“…Together, the data demonstrate that DRS can provide a more comprehensive and bias-free understanding of the T. brucei transcriptome. Provided that the current basecall accuracy and genome-coverage limitations can be overcome [ 40 ], it should be possible to gain further insight into the plasticity of RNA processing reactions in T. brucei , which should accelerate our understanding of the RNA biology of the parasite.…”

Section: Discussionmentioning

confidence: 99%

Nanopore-Based Direct RNA Sequencing of the Trypanosoma brucei Transcriptome Identifies Novel lncRNAs

Kruse

Göringer

2023

Genes

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Trypanosomatids are single-cell eukaryotic parasites. Unlike higher eukaryotes, they control gene expression post-transcriptionally and not at the level of transcription initiation. This involves all known cellular RNA circuits, from mRNA processing to mRNA decay, to translation, in addition to a large panel of RNA-interacting proteins that modulate mRNA abundance. However, other forms of gene regulation, for example by lncRNAs, cannot be excluded. LncRNAs are poorly studied in trypanosomatids, with only a single lncRNA characterized to date. Furthermore, it is not clear whether the complete inventory of trypanosomatid lncRNAs is known, because of the inherent cDNA-recoding and DNA-amplification limitations of short-read RNA sequencing. Here, we overcome these limitations by using long-read direct RNA sequencing (DRS) on nanopore arrays. We analyze the native RNA pool of the two main lifecycle stages of the African trypanosome Trypanosoma brucei, with a special emphasis on the inventory of lncRNAs. We identify 207 previously unknown lncRNAs, 32 of which are stage-specifically expressed. We also present insights into the complexity of the T. brucei transcriptome, including alternative transcriptional start and stop sites and potential transcript isoforms, to provide a bias-free understanding of the intricate RNA landscape in T. brucei.

show abstract

Advances in nanopore direct RNA sequencing

Cited by 79 publications

References 37 publications

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Nanopore-based direct RNA sequencing of theTrypanosoma bruceitranscriptome identifies novel lncRNAs

Nanopore-Based Direct RNA Sequencing of the Trypanosoma brucei Transcriptome Identifies Novel lncRNAs

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