Dataset Information

Comparative analysis of Lactobacillus plantarum WCFS1 transcriptomes by using DNA microarray and next-generation sequencing technologies.

ABSTRACT: RNA sequencing is starting to compete with the use of DNA microarrays for transcription analysis in eukaryotes as well as in prokaryotes. The application of RNA sequencing in prokaryotes requires additional steps in the RNA preparation procedure to increase the relative abundance of mRNA and cannot employ the poly(T)-primed approach in cDNA synthesis. In this study, we aimed to validate the use of RNA sequencing (direct cDNA sequencing and 3'-untranslated region [UTR] sequencing) using Lactobacillus plantarum WCFS1 as a model organism, employing its established microarray platform as a reference. A limited effect of mRNA enrichment on genome-wide transcript quantification was observed, and comparative transcriptome analyses were performed for L. plantarum WCFS1 grown in two different laboratory media. Microarray analyses and both RNA sequencing methods resulted in similar depths of analysis and generated similar fold-change ratios of differentially expressed genes. The highest overall correlation was found between microarray and direct cDNA sequencing-derived transcriptomes, while the 3'-UTR sequencing-derived transcriptome appeared to deviate the most. Overall, a high similarity between patterns of transcript abundance and fold-change levels of differentially expressed genes was detected by all three methods, indicating that the biological conclusions drawn from the transcriptome data were consistent among the three technologies.

SUBMITTER: Leimena MM

PROVIDER: S-EPMC3370542 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Comparative analysis of Lactobacillus plantarum WCFS1 transcriptomes by using DNA microarray and next-generation sequencing technologies.

Leimena Milkha M MM Wels Michiel M Bongers Roger S RS Smid Eddy J EJ Zoetendal Erwin G EG Kleerebezem Michiel M

Applied and environmental microbiology 20120406 12

RNA sequencing is starting to compete with the use of DNA microarrays for transcription analysis in eukaryotes as well as in prokaryotes. The application of RNA sequencing in prokaryotes requires additional steps in the RNA preparation procedure to increase the relative abundance of mRNA and cannot employ the poly(T)-primed approach in cDNA synthesis. In this study, we aimed to validate the use of RNA sequencing (direct cDNA sequencing and 3'-untranslated region [UTR] sequencing) using Lactobaci ...[more]

PMID: 22492454

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Project description:BACKGROUND: Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. RESULTS: Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. CONCLUSIONS: The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants.

Dataset Information

Comparative analysis of Lactobacillus plantarum WCFS1 transcriptomes by using DNA microarray and next-generation sequencing technologies.

Publications

Comparative analysis of Lactobacillus plantarum WCFS1 transcriptomes by using DNA microarray and next-generation sequencing technologies.

Similar Datasets

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