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High-throughput RNA sequencing of pseudomonas-infected Arabidopsis reveals hidden transcriptome complexity and novel splice variants.

ABSTRACT: We report the results of a genome-wide analysis of transcription in Arabidopsis thaliana after treatment with Pseudomonas syringae pathovar tomato. Our time course RNA-Seq experiment uses over 500 million read pairs to provide a detailed characterization of the response to infection in both susceptible and resistant hosts. The set of observed differentially expressed genes is consistent with previous studies, confirming and extending existing findings about genes likely to play an important role in the defense response to Pseudomonas syringae. The high coverage of the Arabidopsis transcriptome resulted in the discovery of a surprisingly large number of alternative splicing (AS) events--more than 44% of multi-exon genes showed evidence for novel AS in at least one of the probed conditions. This demonstrates that the Arabidopsis transcriptome annotation is still highly incomplete, and that AS events are more abundant than expected. To further refine our predictions, we identified genes with statistically significant changes in the ratios of alternative isoforms between treatments. This set includes several genes previously known to be alternatively spliced or expressed during the defense response, and it may serve as a pool of candidate genes for regulated alternative splicing with possible biological relevance for the defense response against invasive pathogens.

SUBMITTER: Howard BE

PROVIDER: S-EPMC3788074 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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High-throughput RNA sequencing of pseudomonas-infected Arabidopsis reveals hidden transcriptome complexity and novel splice variants.

Howard Brian E BE Hu Qiwen Q Babaoglu Ahmet Can AC Chandra Manan M Borghi Monica M Tan Xiaoping X He Luyan L Winter-Sederoff Heike H Gassmann Walter W Veronese Paola P Heber Steffen S

PloS one 20131001 10

We report the results of a genome-wide analysis of transcription in Arabidopsis thaliana after treatment with Pseudomonas syringae pathovar tomato. Our time course RNA-Seq experiment uses over 500 million read pairs to provide a detailed characterization of the response to infection in both susceptible and resistant hosts. The set of observed differentially expressed genes is consistent with previous studies, confirming and extending existing findings about genes likely to play an important role ...[more]

PMID: 24098335

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Project description:BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

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Dataset Information

High-throughput RNA sequencing of pseudomonas-infected Arabidopsis reveals hidden transcriptome complexity and novel splice variants.

Publications

High-throughput RNA sequencing of pseudomonas-infected Arabidopsis reveals hidden transcriptome complexity and novel splice variants.

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