A Comparative Transcriptional Landscape of Two Castor Cultivars Obtained by Single-Molecule Sequencing Comparative Analysis

Background and Objectives: Castor (Ricinus communis L.) is an important non-edible oilseed crop. Lm-type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in their inflorescence structures and leaf shapes. To better understand the mechanisms underlying these differences at the molecular level, we performed a comparative transcriptional analysis.Materials and Methods: Full-length transcriptome sequencing and short-read RNA sequencing were employed.Results: A total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm-type female strains and normal amphiprotic strains, respectively. In Lm-type female strains and normal amphiprotic strains, 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed a great variety of gene expression of common and unique transcription factors between the two cultivars. Meanwhile, a functional analysis of the isoforms was conducted. The full-length sequences were used as a reference genome, and a short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis.Conclusion: The results revealed considerable differences and expression diversity between the two cultivars, well beyond what was reported in previous studies and likely reflecting the differences in architecture between these two cultivars.

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A comparative transcriptional landscape of two castor cultivars obtained by single-molecule sequencing comparative analysis

10.1101/2020.09.30.320101 ◽

2020 ◽

Author(s):

Wei Zhou ◽

Yaxing Zhou ◽

Guoli Zhu ◽

Yun Wang ◽

Zhibiao He ◽

...

Keyword(s):

Transcription Factors ◽

Comparative Analysis ◽

Alternative Splicing ◽

Rna Sequencing ◽

Transcriptome Sequencing ◽

Full Length ◽

Transcriptional Analysis ◽

Sequencing Analysis ◽

Short Read ◽

Alternative Splicing Events

AbstractBackground and ObjectivesCastor (Ricinus communis L.) is an important non-edible oilseed crop. Lm type female strains and normal amphiprotic strains are important castor cultivars, and are mainly different in inflorescence structures and leaf shapes. To better understand the mechanisums underling these differences at the molecular level, we performed comparative transcriptional analysis.Materials and MethodsFull-length transcriptome sequencing and short-read RNA sequencing were employed.ResultsA total of 76,068 and 44,223 non-redundant transcripts were obtained from high-quality transcripts of Lm type female strains and normal amphiprotic strains, respectively. In Lm female strain and normal amphiprotic strains 51,613 and 20,152 alternative splicing events were found, respectively. There were 13,239 transcription factors identified from the full-length transcriptomes. Comparative analysis showed great different gene expression of common and unique transcription factors between the two cultivars. Meanwhile, functional analysis of isoform was conducted. Full-length sequences were used as a reference genome, and short-read RNA sequencing analysis was performed to conduct differential gene analysis. Furthermore, the function of DEGs were performed to annotation analysis.ConclusionsThe results revealed considerable difference and expression diversity between two cultivars, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two cultivars.HighlightUsing the full-length transcriptome sequencing technology, we performed comparative analysis of transcription factors of two castor cultivars, analyzed alternative splicing events, and identified their lncRNAs.

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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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Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method

Communications Biology ◽

10.1038/s42003-020-01125-7 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Yueming Hu ◽

Xing-Sheng Shu ◽

Jiaxian Yu ◽

Ming-an Sun ◽

Zewei Chen ◽

...

Keyword(s):

Rna Sequencing ◽

Single Molecule ◽

Signet Ring Cell ◽

Full Length ◽

Read Length ◽

Accurate Identification ◽

Human Genes ◽

Sequencing Method ◽

Long Read ◽

Gene Expression Studies

AbstractHuman genes form a large variety of isoforms after transcription, encoding distinct transcripts to exert different functions. Single-molecule RNA sequencing facilitates accurate identification of the isoforms by extending nucleotide read length significantly. However, the gene or isoform diversity is lowly represented by the mRNA molecules captured by single-molecule RNA sequencing. Here, we show that a cDNA normalization procedure before the library preparation for PacBio RS II sequencing captures 3.2–6.0 fold more full-length high-quality isoform species for different human samples, as compared to the non-normalized capture procedure. Many lowly expressed, functionally important isoforms can be detected. In addition, normalized PacBio RNA sequencing also resolves more allele-specific haplotype transcripts. Finally, we apply the cDNA normalization based long-read RNA sequencing method to profile the transcriptome of human gastric signet-ring cell carcinomas, identify new cancer-specific transcriptome signatures, and thus, bring out the utility of the improved protocols in gene expression studies.

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Transcriptome Comparative Analysis of Salt Stress Responsiveness in Chrysanthemum (Dendranthema grandiflorum) Roots by Illumina- and Single-Molecule Real-Time-Based RNA Sequencing

DNA and Cell Biology ◽

10.1089/dna.2018.4352 ◽

2018 ◽

Vol 37 (12) ◽

pp. 1016-1030 ◽

Cited By ~ 7

Author(s):

Qian Zhao ◽

Ling He ◽

Bei Wang ◽

Qing-Lin Liu ◽

Yuan-Zhi Pan ◽

...

Keyword(s):

Salt Stress ◽

Comparative Analysis ◽

Real Time ◽

Rna Sequencing ◽

Single Molecule ◽

Dendranthema Grandiflorum ◽

Stress Responsiveness

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A survey of transcriptome complexity using PacBio single-molecule real-time analysis combined with Illumina RNA sequencing for a better understanding of ricinoleic acid biosynthesis in Ricinus communis

BMC Genomics ◽

10.1186/s12864-019-5832-9 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 7

Author(s):

Lijun Wang ◽

Xiaoling Jiang ◽

Lei Wang ◽

Wei Wang ◽

Chunling Fu ◽

...

Keyword(s):

Real Time ◽

Rna Sequencing ◽

Ricinoleic Acid ◽

Single Molecule ◽

Ricinus Communis ◽

Time Analysis ◽

Acid Biosynthesis ◽

Real Time Analysis

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Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer

Genome Biology ◽

10.1186/s13059-021-02261-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Kie Kyon Huang ◽

Jiawen Huang ◽

Jeanie Kar Leng Wu ◽

Minghui Lee ◽

Su Ting Tay ◽

...

Keyword(s):

Gastric Cancer ◽

Rna Sequencing ◽

Molecular Subtypes ◽

Full Length ◽

Sequencing Data ◽

Gastrointestinal Malignancies ◽

Coding Sequences ◽

Short Read ◽

Long Read ◽

Novel Isoforms

Abstract Background Deregulated gene expression is a hallmark of cancer; however, most studies to date have analyzed short-read RNA sequencing data with inherent limitations. Here, we combine PacBio long-read isoform sequencing (Iso-Seq) and Illumina paired-end short-read RNA sequencing to comprehensively survey the transcriptome of gastric cancer (GC), a leading cause of global cancer mortality. Results We performed full-length transcriptome analysis across 10 GC cell lines covering four major GC molecular subtypes (chromosomal unstable, Epstein-Barr positive, genome stable and microsatellite unstable). We identify 60,239 non-redundant full-length transcripts, of which > 66% are novel compared to current transcriptome databases. Novel isoforms are more likely to be cell line and subtype specific, expressed at lower levels with larger number of exons, with longer isoform/coding sequence lengths. Most novel isoforms utilize an alternate first exon, and compared to other alternative splicing categories, are expressed at higher levels and exhibit higher variability. Collectively, we observe alternate promoter usage in 25% of detected genes, with the majority (84.2%) of known/novel promoter pairs exhibiting potential changes in their coding sequences. Mapping these alternate promoters to TCGA GC samples, we identify several cancer-associated isoforms, including novel variants of oncogenes. Tumor-specific transcript isoforms tend to alter protein coding sequences to a larger extent than other isoforms. Analysis of outcome data suggests that novel isoforms may impart additional prognostic information. Conclusions Our results provide a rich resource of full-length transcriptome data for deeper studies of GC and other gastrointestinal malignancies.

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A gene toolbox for monitoring autophagy transcription

Cell Death and Disease ◽

10.1038/s41419-021-04121-9 ◽

2021 ◽

Vol 12 (11) ◽

Author(s):

Matteo Bordi ◽

Rossella De Cegli ◽

Beatrice Testa ◽

Ralph A. Nixon ◽

Andrea Ballabio ◽

...

Keyword(s):

Transcription Factors ◽

Rna Sequencing ◽

Real Time Pcr ◽

Cellular Localization ◽

Gene List ◽

Sequencing Analysis ◽

Functional Protein ◽

Mtor Inhibition ◽

Step Process ◽

Autophagy Pathway

AbstractAutophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the “induction list” by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps.

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Full-coverage native RNA sequencing of HIV-1 viruses

10.1101/845610 ◽

2019 ◽

Author(s):

Alejandro R. Gener ◽

Jason T. Kimata

Keyword(s):

Rna Sequencing ◽

Single Molecule ◽

Transcriptional Start Site ◽

Full Length ◽

Context Dependency ◽

Rna Modification ◽

Clinical Samples ◽

Single Nucleotide Variants ◽

Sequence Alignments ◽

Hiv 1

ABSTRACTObjectiveTo evaluate native RNA sequencing for sequencing HIV-1 viral genomesMethodsFifteen HIV-1 strains were processed with Direct RNA Sequencing (SQK-RNA002) library kits and sequenced on MinION Mk1B devices with RevD flow cells (Oxford Nanopore Technologies (ONT), Oxford, UK). Raw reads were converted to FASTQ, aligned to reference sequences, and assembled into contigs. Multi-sequence alignments of the contigs were generated and used for cladistics analysis.ResultsWe sequenced full-length HIV-1 from the transcriptional start site to 3’ LTR (100% virion genome) in 3 out of 15 isolates (89.6, NLAD8, AD17), achieving majority coverage (defined as > 50%) in another 7 out of 15 isolates. Inspection of NLAD8 sequence alignments revealed splicing or deletion signatures. Despite the strong 3’ bias, read coverage was sufficient to evaluate single-nucleotide variants (SNVs), insertions and deletions in 9 isolates, and to assemble HIV-1 genomes directly from viral RNA, achieving a maximum of 94% assembly coverage for NLAD8. Phylogenetic relationships were maintained at the level of contigs, as well as individual reads.ConclusionsONT native RNA sequencing performed as expected, covering full-length HIV-1 RNA without PCR or cDNA sequencing. Native single-molecule RNA sequencing supported previous models of HIV-1 replication, and samples exhibited strain-specific transcriptional signals. We propose Context Dependency Variant Classification to describe variants occurring in information-dense regions of HIV. These data provide rich resources for emerging RNA modification detection schemes. Future work will expand HIV-1 transcript profiling to infection models and clinical samples.

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