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Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

Zywicki M, Bakowska-Zywicka K, Polacek N - Nucleic Acids Res. (2012)

Bottom Line: Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases.Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data.To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and RNomics, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria. marek.zywicki@i-med.ac.at

ABSTRACT
The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

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A summary of the genomic features identified in the ribosome-derived cDNA library. As indicated, for most of the contigs putative processing products were observed.
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gks020-F3: A summary of the genomic features identified in the ribosome-derived cDNA library. As indicated, for most of the contigs putative processing products were observed.

Mentions: To highlight all the above mentioned features, we have applied APART to deep-sequencing data of a specialized S. cerevisiae cDNA library. We have generated a cDNA library from small RNAs (sized 20–500 nt) that co-purify with ribosomes under different environmental conditions. After sequencing, we have obtained a pool of 125 868 raw reads containing amplification adapters on both the 5′- and 3′-ends and an additional poly-C-tail at the 3′-end which has been used for initial RT [‘Materials and Methods’ section and (6)]. During the cleaning procedure, 81 790 reads were discarded due to length exclusions (<18 nt) or due to the read quality filters. For downstream analysis, we have used 18 679 reads for which adapters on both ends could be detected to ensure that all the reads are derived from full-length cellular RNAs. In total, 12 494 reads (66.89%) were mapped to the reference yeast genome with a maximum of 100 genomic hits. These reads were assembled into 716 contigs each containing at least two reads. After read name-based clustering, we have obtained 174 representative contigs. For 131 of those, the APART pipeline has detected at least one possible stable RNA species. Most of them were processing products derived predominantly from rRNAs, tRNAs and snoRNAs (Figure 3).Figure 3.


Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

Zywicki M, Bakowska-Zywicka K, Polacek N - Nucleic Acids Res. (2012)

A summary of the genomic features identified in the ribosome-derived cDNA library. As indicated, for most of the contigs putative processing products were observed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351166&req=5

gks020-F3: A summary of the genomic features identified in the ribosome-derived cDNA library. As indicated, for most of the contigs putative processing products were observed.
Mentions: To highlight all the above mentioned features, we have applied APART to deep-sequencing data of a specialized S. cerevisiae cDNA library. We have generated a cDNA library from small RNAs (sized 20–500 nt) that co-purify with ribosomes under different environmental conditions. After sequencing, we have obtained a pool of 125 868 raw reads containing amplification adapters on both the 5′- and 3′-ends and an additional poly-C-tail at the 3′-end which has been used for initial RT [‘Materials and Methods’ section and (6)]. During the cleaning procedure, 81 790 reads were discarded due to length exclusions (<18 nt) or due to the read quality filters. For downstream analysis, we have used 18 679 reads for which adapters on both ends could be detected to ensure that all the reads are derived from full-length cellular RNAs. In total, 12 494 reads (66.89%) were mapped to the reference yeast genome with a maximum of 100 genomic hits. These reads were assembled into 716 contigs each containing at least two reads. After read name-based clustering, we have obtained 174 representative contigs. For 131 of those, the APART pipeline has detected at least one possible stable RNA species. Most of them were processing products derived predominantly from rRNAs, tRNAs and snoRNAs (Figure 3).Figure 3.

Bottom Line: Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases.Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data.To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Innsbruck Biocenter, Medical University Innsbruck, Division of Genomics and RNomics, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria. marek.zywicki@i-med.ac.at

ABSTRACT
The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

Show MeSH
Related in: MedlinePlus