High-Resolution Transcriptome Analysis with Long-Read RNA Sequencing

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.

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Faculty Opinions recommendation of Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725503882.793524932 ◽

2016 ◽

Author(s):

Auinash Kalsotra

Keyword(s):

Transcriptome Analysis ◽

Long Read

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Comparative transcriptome analysis of thiamethoxam susceptible and resistant Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), using RNA‐sequencing

Insect Science ◽

10.1111/1744-7917.12901 ◽

2021 ◽

Author(s):

Xue Dong Chen ◽

Surendra Neupane ◽

Torrence A. Gill ◽

Hunter Gossett ◽

Kirsten S. Pelz‐Stelinski ◽

...

Keyword(s):

Rna Sequencing ◽

Transcriptome Analysis ◽

Asian Citrus Psyllid ◽

Diaphorina Citri ◽

Comparative Transcriptome ◽

Comparative Transcriptome Analysis

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Tumor Heterogeneity Regarding Radiosensitivity, Recurrence Risk, and Immune-Checkpoint in Breast Cancer: Transcriptome Analysis of Single-cell RNA Sequencing Data

International Journal of Radiation Oncology*Biology*Physics ◽

10.1016/j.ijrobp.2019.06.1062 ◽

2019 ◽

Vol 105 (1) ◽

pp. E664

Author(s):

B.S. Jang ◽

W. Han ◽

I.A. Kim

Keyword(s):

Breast Cancer ◽

Single Cell ◽

Rna Sequencing ◽

Transcriptome Analysis ◽

Immune Checkpoint ◽

Tumor Heterogeneity ◽

Recurrence Risk ◽

Sequencing Data ◽

Single Cell Rna Sequencing ◽

Cancer Transcriptome

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High resolution species detection: accurate long read eDNA metabarcoding of North Sea fish using Oxford Nanopore sequencing

10.22541/au.163828284.40261296/v1 ◽

2021 ◽

Author(s):

Karlijn Doorenspleet ◽

Lara Jansen ◽

Saskia Oosterbroek ◽

Oscar Bos ◽

Pauline Kamermans ◽

...

Keyword(s):

High Resolution ◽

North Sea ◽

Fish Species ◽

16S Rrna Genes ◽

Rrna Genes ◽

Nanopore Sequencing ◽

Nature Restoration ◽

Oxford Nanopore ◽

Field Samples ◽

Long Read

To monitor the effect of nature restoration projects in North Sea ecosystems, accurate and intensive biodiversity assessments are vital. DNA based techniques and especially environmental DNA (eDNA) metabarcoding from seawater is becoming a powerful monitoring tool. However, current approaches are based on genetic target regions of <500 nucleotides, which offer limited taxonomic resolution. This study aims to develop and validate a long read nanopore sequencing method for eDNA that enables improved identification of fish species. We designed a universal primer pair targeting a 2kb region covering the 12S and 16S rRNA genes of fish mitochondria. eDNA was amplified and sequenced using the Oxford Nanopore MiniON. Sequence data was processed using the new pipeline Decona, and accurate consensus identities of above 99.9% were retrieved. The primer set efficiency was tested with eDNA from a 3.000.000 L zoo aquarium with 31 species of bony fish and elasmobranchs. Over 55% of the species present were identified on species level and over 75% on genus level. Next, our long read eDNA metabarcoding approach was applied to North Sea eDNA field samples collected at ship wreck sites, the Gemini Offshore Wind Farm, the Borkum Reef Grounds and a bare sand bottom. Here, location specific fish and vertebrate communities were obtained. Incomplete reference databases still form a major bottleneck in further developing high resolution long read metabarcoding. Yet, the method has great potential for rapid and accurate fish species monitoring in marine field studies.

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TagSeqTools: a flexible and comprehensive analysis pipeline for NAD tagSeq data

10.1101/2020.03.09.982934 ◽

2020 ◽

Cited By ~ 1

Author(s):

Huan Zhong ◽

Zongwei Cai ◽

Zhu Yang ◽

Yiji Xia

Keyword(s):

Rna Sequencing ◽

Comprehensive Analysis ◽

Enzymatic Reactions ◽

Computational Tool ◽

Sequencing Data ◽

Analysis Pipeline ◽

Oxford Nanopore ◽

Long Read ◽

Identification And Characterization

AbstractNAD tagSeq has recently been developed for the identification and characterization of NAD+-capped RNAs (NAD-RNAs). This method adopts a strategy of chemo-enzymatic reactions to label the NAD-RNAs with a synthetic RNA tag before subjecting to the Oxford Nanopore direct RNA sequencing. A computational tool designed for analyzing the sequencing data of tagged RNA will facilitate the broader application of this method. Hence, we introduce TagSeqTools as a flexible, general pipeline for the identification and quantification of tagged RNAs (i.e., NAD+-capped RNAs) using long-read transcriptome sequencing data generated by NAD tagSeq method. TagSeqTools comprises two major modules, TagSeek for differentiating tagged and untagged reads, and TagSeqQuant for the quantitative and further characterization analysis of genes and isoforms. Besides, the pipeline also integrates some advanced functions to identify antisense or splicing, and supports the data reformation for visualization. Therefore, TagSeqTools provides a convenient and comprehensive workflow for researchers to analyze the data produced by the NAD tagSeq method or other tagging-based experiments using Oxford nanopore direct RNA sequencing. The pipeline is available at https://github.com/dorothyzh/TagSeqTools, under Apache License 2.0.

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The architecture of SARS-CoV-2 transcriptome

10.1101/2020.03.12.988865 ◽

2020 ◽

Cited By ~ 38

Author(s):

Dongwan Kim ◽

Joo-Yeon Lee ◽

Jeong-Sun Yang ◽

Jun Won Kim ◽

V. Narry Kim ◽

...

Keyword(s):

High Resolution ◽

Rna Sequencing ◽

Rna Modification ◽

List Type ◽

Rna Modifications ◽

Subgenomic Rnas ◽

Frequent Motif ◽

High Resolution Map ◽

Functional Investigation ◽

Resolution Map

AbstractSARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3′ tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.HighlightsWe provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing.The transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical.In addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs.We discover at least 41 potential RNA modification sites with an AAGAA motif.

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