Friday, November 18, 2011

Ribosomal RNA depletion produces improved whole-transcriptome RNA-Seq results

In a recent PLoSOne publication, Huang et al. compared the use of the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) to the RiboMinus™ rRNA Removal Kit (Life Technologies) and poly(A) enrichment in preparation of RNA-Seq libraries. They studied two types of mouse tissue samples: differentiated embryonic stem cells (CCE) and fetal head (FH).
The researchers compared samples that were treated by rRNA removal and RNA chemical fragmentation procedures on the basis of gene expression analysis, quality of sequence data, number of reads, retention of sequence tags, and any biases noted in the sequencing. Some notable conclusions from this study:
  1. RiboMinus treatment may require two rounds of rRNA depletion whereas Ribo-Zero treatment only requires round of depletion.
  2. The use of RNA hydrolysis and Ribo-Zero treatment result in more efficient removal of rRNA, as compared to RNA hydrolysis and RiboMinus treatment. The researchers also observed an increase in uniquely matching (36%-54%) and multiple matching tags (28%-30%). Interestingly, tags with no match in the genome did not change (14%-21%.)
  3. In comparison to the RiboMinus preparation, Ribo-Zero treatment results in RNA-Seq libraries with more tags in the 5′ and 3′ ends of small genes compared to the body of the genes. The coverage of other gene sizes was largely unchanged between the two rRNA depletion methods.
  4. RNA-Seq  libraries prepared from ribo-depleted (e.g., Ribo-Zero-treated) RNA have significant advantages over poly(A)-enriched RNA for detecting macro ncRNAs.
ResearchBlogging.orgHuang, R. 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) DOI: 10.1371/journal.pone.0027288

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