Jun

27

NorahDesk – a small non-coding RNA (ncRNA) detection tool for RNA sequencing (RNA-Seq) data

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NorahDeskNorahDesk reconstructs full-length putative ncRNA transcripts from short sequence reads by hybridizing contigs. It analyzes not only the distinct read distribution of true ncRNA classes in an unbiased way but also utilizes secondary structures as an independent confirmation source to reliably predict ncRNA from deep sequencing data.

Using publicly available mouse sequence data from brain, skeletal muscle, testis and ovary, NorahDesk was evaluated with an emphasis on the performance for microRNAs (miRNAs) and piwi-interacting small RNA (piRNA). This method was also compared with Dario and mirDeep2 and found to produce longer transcripts with higher read coverage. This feature makes it the first method particularly suitable for the prediction of both known and novel piRNAs.

NorahDesk and the mouse small ncRNA annotation file in BED format used in this study are available at http://www.bioinformatics.org.au/NorahDesk.

  • Ragan C, Mowry BJ, Bauer DC. (2012) Hybridization-based reconstruction of small non-coding RNA transcripts from deep sequencing data. Nucleic Acids Res [Epub ahead of print]. [article]

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Nov

22

New RNA-Seq template preparation protocol enables detection of imprinted macro ncRNAs

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A team led by researchers at the Austrian Academy of Sciences has developed a protocol that includes column-free RNA preparation, ribosomal RNA depletion, RNA-hydrolysis and ds-cDNA synthesis and is compatible with the all of the major NGS platforms used to date.

They demonstrate that:

  • Ribo-depleted RNA-Seq is highly reproducible between different sequencing locations, even when using two different ribosomal RNA depletion strategies.
  • Template fragmentation by RNA-hydrolysis produces more homogenous gene coverage than cDNA shearing, and that both fragmentation methods lead to under-representation of 5′ and 3′ UTRs.
  • The use of similar template preparation protocols is critical for obtaining a comparable transcriptome.
  • RNA populations prepared by ribo-depletion allow RNA-Seq to reliably detect both the non-coding and protein-coding transcriptome, and also to identify biologically relevant gene expression differences in both of these RNA types.

Huang R, Jaritz M, Guenzl P, Vlatkovic I, Sommer A, et al. (2011) An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs. PLoS ONE 6(11), e27288. [article]

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Aug

25

NIH-Oxford collaboration uses RNA-Seq to produce detailed map of gene activity in mouse brain

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brain activityThe researchers used a new sequencing technology called RNA-seq, a technology related to the latest DNA sequencers used to decode our genomes, to map gene activity in the different layers of the mouse cerebral cortex.

The technique is also able to detect ‘non-coding RNAs’, ie RNAs produced from DNA in between known genes that doesn’t code for proteins but may play a critical role in regulating genes and controlling biological processes.

‘We see a vast array of non-coding RNAs – hundreds that have never been seen before, but presumably have a biological role to play in the brain,’ says Professor Molnár. ‘One of the most abundant RNAs produced in the mouse brain is a noncoding RNA.’

The approach also reveals RNAs which, once read off from our DNA code, are stitched together in different ways through a process called ‘alternative splicing’. The process results in different proteins that can have different biological roles, despite coming from the same gene.

NIH Press Release

Oxford University Press Release

  • T. Grant Belgard, Ana C. Marques, Peter L. Oliver, Hatice Ozel Abaan, Tamara M. Sirey, Anna Hoerder-Suabedissen, Fernando García-Moreno, Zoltán Molnár, Elliott H. Margulies, Chris P. Ponting (2011) A Transcriptomic Atlas of Mouse Neocortical Layers. Neuron 71(4),  605-16. [abstract]

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Oct

5

Protein Regulation Mechanism Differs by RNA Length

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Thanks to the new technological advances in genome and transcriptome sequencing, we know that a major portion of the genome is being transcribed but only a small portion of this transcriptome contains the protein-coding sequences. The remainder can be split (at approximately 100 nucleotide length) into two major categories: small non-coding RNA (including microRNA) and long non-coding RNA, both of which have been shown to exert regulatory control over protein production/expression. 

Interestingly, these two types of non-coding RNA exert their control by very different mechanisms.  Small RNAs regulate gene expression predominantly through reduction of mRNA levels and subsequent reduced protein output1 (negative regulation), while long non-coding RNAs increase the synthesis of nearby proteins2 (positive regulation).

(read more about negative regulation by small non-coding RNAs… )

(read more about positive regulation by long non-coding RNAs… ) 

  1. Guo H, Ingolia NT, Weissman JS, Bartel DP. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466(7308), 835-40. [abstract]
  2. Orom UA, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G, Lai F, Zytnicki M, Notredame C, Huang Q, Guigo R, Shiekhattar R. (2010) Long Noncoding RNAs with Enhancer-like Function in Human Cells. Cell143(1), 46-58. [abstract]

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  • RSS SEQanswers – RNA Sequencing

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