Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing

Zhao, Liangzhen; Zhang, Hangxiao; Kohnen, Markus V.; Prasad, Kasavajhala V. S. K.; Gu, Lianfeng; Reddy, Anireddy S. N.

doi:10.3389/fgene.2019.00253

Cited by 139 publications

(97 citation statements)

References 137 publications

(206 reference statements)

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“…Future work will aim to extend our analyses to larger numbers of samples to explore population-level variation in transcript abundance in the cerebral cortex and differences associated with pathology. Second, despite the advantages of long-read sequencing approaches for the characterization of novel full-length transcripts, these methods are often assumed to be less quantitative than traditional short-read RNA sequencing methods 66 . We implemented a stringent QC pipeline (see Methods) and undertook considerable filtering of our data, finding high consistency across biological replicates and validating our findings using complementary approaches (i.e.…”

Section: Discussionmentioning

confidence: 99%

Full-length transcript sequencing of human and mouse identifies widespread isoform diversity and alternative splicing in the cerebral cortex

Jeffries

Leung

Castanho

et al. 2020

Preprint

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Alternative splicing is a post-transcriptional regulatory mechanism producing multiple distinct mRNA molecules from a single pre-mRNA. Alternative splicing has a prominent role in the central nervous system, impacting neurodevelopment and various neuronal functions as well as being increasingly implicated in brain disorders including autism, schizophrenia and Alzheimer's disease. Standard short-read RNA-Seq approaches only sequence fragments of the mRNA molecule, making it difficult to accurately characterize the true nature of RNA isoform diversity. In this study, we used long-read isoform sequencing (Iso-Seq) to generate full-length cDNA sequences and map transcript diversity in the human and mouse cerebral cortex. We identify widespread RNA isoform diversity amongst expressed genes in the cortex, including many novel transcripts not present in existing genome annotations. Alternative splicing events were found to make a major contribution to RNA isoform diversity in the cortex, with intron retention being a relatively common event associated with nonsense-mediated decay and reduced transcript expression. Of note, we found evidence for transcription from novel (unannotated genes) and fusion events between neighbouring genes. Although global patterns of RNA isoform diversity were found to be generally similar between human and mouse cortex, we identified some notable exceptions. We also identified striking developmental changes in transcript diversity, with differential transcript usage between human adult and fetal cerebral cortex. Finally, we found evidence for extensive isoform diversity in genes associated with autism, schizophrenia and Alzheimer's disease. Our data confirm the importance of alternative splicing in the cerebral cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide this transcript level data as a resource to the scientific community.

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Section: Discussionmentioning

confidence: 99%

Full-length transcript sequencing of human and mouse identifies widespread isoform diversity and alternative splicing in the cerebral cortex

Jeffries

Leung

Castanho

et al. 2020

Preprint

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“…One weakness of nanopore sequencing is its low accuracy that leads to false negatives including false alternative isoforms and splicing events. FLAIR and other long read tools require reads correction to compensate for nanopore low accuracy (Zhao et al 2019). But UNAGI filter out these false events based on long reads themselves.…”

Section: Discussionmentioning

confidence: 99%

“…For short reads, the Tuxedo package, which is among the most popular tools, performs the reconstruction using Cufflinks or its successor StringTie. For long reads, transcriptome assembly is done either de novo without a reference or de novo with the guidance of a reference genome in case of its presence (Zhao et al 2019). Tools for genome-guided transcriptome reconstruction using long reads are designed primarily for Pac Bio long reads and not ONT reads (Bayega et al 2018).…”

Section: Introductionmentioning

confidence: 99%

UNAGI: an automated pipeline for nanopore full-length cDNA sequencing uncovers novel transcripts and isoforms in yeast

Kadi

Jung

Ito

et al. 2020

Funct Integr Genomics

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Sequencing the entire RNA molecule leads to a better understanding of the transcriptome architecture. SMARTer (Switching Mechanism at 5′-End of RNA Template) is a technology aimed at generating full-length cDNA from low amounts of mRNA for sequencing by short-read sequencers such as those from Illumina. However, short read sequencing such as Illumina technology includes fragmentation that results in bias and information loss. Here, we built a pipeline, UNAGI or UNAnnotated Gene Identifier, to process long reads obtained with nanopore sequencing and compared this pipeline with the standard Illumina pipeline by studying the Saccharomyces cerevisiae transcriptome in full-length cDNA samples generated from two different biological samples: haploid and diploid cells. Additionally, we processed the long reads with another long read tool, FLAIR. Our strand-aware method revealed significant differential gene expression that was masked in Illumina data by antisense transcripts. Our pipeline, UNAGI, outperformed the Illumina pipeline and FLAIR in transcript reconstruction (sensitivity and specificity of 80% and 40% vs. 18% and 34% and 79% and 32%, respectively). Moreover, UNAGI discovered 3877 unannotated transcripts including 1282 intergenic transcripts while the Illumina pipeline discovered only 238 unannotated transcripts. For isoforms profiling, UNAGI also outperformed the Illumina pipeline and FLAIR in terms of sensitivity (91% vs. 82% and 63%, respectively). But the low accuracy of nanopore sequencing led to a closer gap in terms of specificity with Illumina pipeline (70% vs. 63%) and to a huge gap with FLAIR (70% vs 0.02%).

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“…Based on the PacBio and Illumina sequencing data, we identi ed 8,139 DETs from 6,577 DEGs in M. sieversii during the response to the infection of C. mali. In eukaryotes, the APA and AS events are the major processes that contribute to transcriptome diversity [29,34]. A total of 11,733 isoforms in Oryza sativa L. [35], 110,00 non-redundant isoforms in Zea mays [36], and 29,730 novel isoforms in Trifolium pratense L. [37] were identi ed using Pacbio sequencing.…”

Section: Discussionmentioning

confidence: 99%

PacBio full-length transcriptome of wild apple (Malus sieversii) provides insights into canker disease dynamic response

Liu

Wen

et al. 2020

Preprint

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Background Cytospora canker is a serious disease in the stem of Malus sieversii, caused by Cytospora mali. However, little is known about the global response mechanism in M. sieversii to C. mali infection. Results Phytohormone jasmonic acid (JA) and salicylic acid (SA) profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that the JA was initially produced to respond to the necrotrophic pathogen C. mali infection at the early response stage, then get synergistically transduced with SA to respond at the late response stage. Furthermore, we adopted Pacific Biosciences (PacBio) full-length sequencing to identify differentially expressed transcripts (DETs) during the canker response stage. We obtained 52,538 full-length transcripts, of which 8,139 were DETs. Total 1,336 lncRNAs, 23,737 alternative polyadenylation (APA) sites and 3,780 putative transcription factors (TFs) were identified. Additionally, functional annotation analysis of DETs indicated that the wild apple response to the infection of C. mali involves plant-pathogen interaction, plant hormone signal transduction, flavonoid biosynthesis, and phenylpropanoid biosynthesis. The co-expression network of the differentially expressed TFs revealed 264 candidate TF transcripts. Among these candidates, the WRKY family was the most abundant. The MsWRKY7 and MsWRKY33 were highly correlated at the early response stage, and MsWRKY6, MsWRKY7, MsWRKY19, MsWRKY33, MsWRKY40, MsWRKY45, MsWRKY51, MsWRKY61, MsWRKY75 were highly correlated at the late stage. Conclusions The full-length transcriptomic analysis revealed a series of immune responsive events in M. sieversii in response to C. mali infection. The phytohormone signal pathway regulatory played an important role in the response stage. Additionally, the enriched disease resistance pathways and differentially expressed TFs dynamics collectively contributed to the immune response. This study provides valuable insights into a dynamic response in M. sieversii upon the necrotrophic pathogen C. mali infection, facilitates understanding of response mechanisms to canker disease for apple, and provides supports in the identification of potential resistance genes in M. sieversii.

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Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing

Cited by 139 publications

References 137 publications

Full-length transcript sequencing of human and mouse identifies widespread isoform diversity and alternative splicing in the cerebral cortex

Full-length transcript sequencing of human and mouse identifies widespread isoform diversity and alternative splicing in the cerebral cortex

UNAGI: an automated pipeline for nanopore full-length cDNA sequencing uncovers novel transcripts and isoforms in yeast

PacBio full-length transcriptome of wild apple (Malus sieversii) provides insights into canker disease dynamic response

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