Transcriptome Profiling Using Single-Molecule Direct RNA Sequencing

Background: The standard approach for transcriptomic profiling involves high throughput short-read sequencing technology, mainly dominated by Illumina. However, the short reads have limitations in transcriptome assembly and in obtaining full-length transcripts due to the complex nature of transcriptomes with variable length and multiple alternative spliced isoforms. Recent advances in long read sequencing by the Oxford Nanopore Technologies (ONT) offered both cDNA as well as direct RNA sequencing and has brought a paradigm change in the sequencing technology to greatly improve the assembly and expression estimates. ONT enables molecules to be sequenced without fragmentation resulting in ultra-long read length enabling the entire genes and transcripts to be fully characterized. The direct RNA sequencing method, in addition, circumvents the reverse transcription and amplification steps. Objective: In this study, RNA sequencing methods were assessed by comparing data from Illumina (ILM), ONT cDNA (OCD) and ONT direct RNA (ODR). Methods: The sensitivity & specificity of the isoform detection was determined from the data generated by Illumina, ONT cDNA and ONT direct RNA sequencing technologies using Saccharomyces cerevisiae as model. Comparative studies were conducted with two pipelines to detect the isoforms, novel genes and variable gene length. Results: Mapping metrics and qualitative profiles for different pipelines are presented to understand these disruptive technologies. The variability in sequencing technology and the analysis pipeline were studied.

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Faculty Opinions recommendation of MicroRNA transcriptome profiling of mice brains infected with Japanese encephalitis virus by RNA sequencing.

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

10.3410/f.726121839.793528999 ◽

2017 ◽

Author(s):

Anirban Basu

Keyword(s):

Japanese Encephalitis Virus ◽

Rna Sequencing ◽

Japanese Encephalitis ◽

Transcriptome Profiling ◽

Encephalitis Virus

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Understanding Disease Mechanisms of Hereditary Angioedema With Potential Therapeutic Implications Through RNA Sequencing Transcriptome Profiling

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2020.12.113 ◽

2021 ◽

Vol 147 (2) ◽

pp. AB20

Author(s):

Umesh Singh ◽

Debayoti Ghosh ◽

Jonathan Bernstein

Keyword(s):

Rna Sequencing ◽

Hereditary Angioedema ◽

Transcriptome Profiling ◽

Therapeutic Implications ◽

Disease Mechanisms

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Single molecule real time (SMRT) full length RNA-sequencing reveals novel and distinct mRNA isoforms in human bone marrow cell subpopulations

10.1101/555326 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anne Deslattes Mays ◽

Marcel O. Schmidt ◽

Garrett T. Graham ◽

Elizabeth Tseng ◽

Primo Baybayan ◽

...

Keyword(s):

Bone Marrow ◽

Real Time ◽

Rna Sequencing ◽

Single Molecule ◽

Marrow Cell ◽

Full Length ◽

Protein Isoforms ◽

Mrna Isoforms ◽

Transcript Isoforms ◽

Cell Subpopulations

AbstractHematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single molecule, real time (SMRT) full length RNA sequencing. This analysis revealed a ∼5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of mRNA isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was validated by mass spectrometry and validated previously unknown protein isoforms predicted e.g. for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g. CFD, GATA2, HLA-A, B & C) also distinguished cell subpopulations but were only detectable by full length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

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Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification

eLife ◽

10.7554/elife.49658 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 43

Author(s):

Matthew T Parker ◽

Katarzyna Knop ◽

Anna V Sherwood ◽

Nicholas J Schurch ◽

Katarzyna Mackinnon ◽

...

Keyword(s):

Rna Sequencing ◽

Single Molecule ◽

Tail Length ◽

Transcript Abundance ◽

Mrna Processing ◽

Full Length ◽

Transcription Start Sites ◽

Model Plant Arabidopsis Thaliana ◽

Defective Rna ◽

A Site

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

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A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages

European Respiratory Journal ◽

10.1183/13993003.00646-2019 ◽

2019 ◽

Vol 55 (1) ◽

pp. 1900646 ◽

Cited By ~ 30

Author(s):

Nikita Joshi ◽

Satoshi Watanabe ◽

Rohan Verma ◽

Renea P. Jablonski ◽

Ching-I Chen ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Single Cell ◽

Rna Sequencing ◽

Single Molecule ◽

Alveolar Macrophages ◽

Lineage Tracing ◽

Single Cell Rna Sequencing ◽

Pharmacological Blockade ◽

Macrophage Colony ◽

Druggable Targets

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

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3′ MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage

Laboratory Investigation ◽

10.1038/s41374-020-0446-z ◽

2020 ◽

Vol 100 (10) ◽

pp. 1345-1355 ◽

Cited By ~ 2

Author(s):

Stefaniya Boneva ◽

Anja Schlecht ◽

Daniel Böhringer ◽

Hans Mittelviefhaus ◽

Thomas Reinhard ◽

...

Keyword(s):

Rna Sequencing ◽

Storage Time ◽

Transcriptome Profiling ◽

Rna Seq ◽

Fresh Tissue ◽

Long Term Storage ◽

Ffpe Samples ◽

The Impact ◽

Term Storage

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

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Transcriptome Profiling Provides Insight into the Genes in Carotenoid Biosynthesis during the Mesocarp and Seed Developmental Stages of Avocado (Persea americana)

International Journal of Molecular Sciences ◽

10.3390/ijms20174117 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4117 ◽

Cited By ~ 8

Author(s):

Yu Ge ◽

Zhihao Cheng ◽

Xiongyuan Si ◽

Weihong Ma ◽

Lin Tan ◽

...

Keyword(s):

Single Molecule ◽

Developmental Stages ◽

Gene Dosage ◽

Average Length ◽

Beta Carotene ◽

Transcriptome Profiling ◽

Carotenoid Biosynthesis ◽

Persea Americana ◽

Sequencing Data ◽

Long Read

Avocado (Persea americana Mill.) is an economically important crop because of its high nutritional value. However, the absence of a sequenced avocado reference genome has hindered investigations of secondary metabolism. For next-generation high-throughput transcriptome sequencing, we obtained 365,615,152 and 348,623,402 clean reads as well as 109.13 and 104.10 Gb of sequencing data for avocado mesocarp and seed, respectively, during five developmental stages. High-quality reads were assembled into 100,837 unigenes with an average length of 847.40 bp (N50 = 1725 bp). Additionally, 16,903 differentially expressed genes (DEGs) were detected, 17 of which were related to carotenoid biosynthesis. The expression levels of most of these 17 DEGs were higher in the mesocarp than in the seed during five developmental stages. In this study, the avocado mesocarp and seed transcriptome were also sequenced using single-molecule long-read sequencing to acquired 25.79 and 17.67 Gb clean data, respectively. We identified 233,014 and 238,219 consensus isoforms in avocado mesocarp and seed, respectively. Furthermore, 104 and 59 isoforms were found to correspond to the putative 11 carotenoid biosynthetic-related genes in the avocado mesocarp and seed, respectively. The isoform numbers of 10 out of the putative 11 genes involved in the carotenoid biosynthetic pathway were higher in the mesocarp than those in the seed. Besides, alpha- and beta-carotene contents in the avocado mesocarp and seed during five developmental stages were also measured, and they were higher in the mesocarp than in the seed, which validated the results of transcriptome profiling. Gene expression changes and the associated variations in gene dosage could influence carotenoid biosynthesis. These results will help to further elucidate carotenoid biosynthesis in avocado.

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