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

Wednesday, November 9, 2011

Medicinal leeches, MasterPure RNA Kits, and metatranscriptomics

The overwhelming majority of microorganisms in existence have not been cultured in the laboratory. Even the most abundant and metabolically active members of the microbial community can be challenging to cultivate. Next-generation sequencing is providing insight into the metabolic relationships within microbial communities by sequencing the metatranscriptome.

Bomar et al. report on the gut microbiome of the medicinal leech Hirudo verbena. They found two symbionts in the digestive tract: i) a Rikenella-like bacterium; and ii) Aeromonas veronii. After a blood meal, the largest compartment of the digestive tract (the crop), stores ingested blood for months between feedings. Water and salts are removed from the ingested blood meal, creating a viscous intraluminal fluid (ILF) that contains these symbiotic bacteria. RNA from the ILF was extracted using the MasterPure™ RNA Purification Kit. After mRNA enrichment, an Illumina cDNA library was constructed. Approximatley 81% of the reads mapped to A. veronii or Rikenella. The Rikenella-like bacterium’s transcriptome indicated that energy is obtained by fermenting sugars to acetate. Surprisingly, the Rikenella-like symbiont foraged host mucin glycans rather than the blood meal. Its capacity to use mucins may be important for its ability to survive within the leech gut for up to 6 months between feedings. The discovery of mucin as the primary nutrient enabled the design of a medium that allowed the cultivation of Rikenella under conditions that favored its growth, and pure cultures were obtained. Thus, the application of metatranscriptomics to growing bacteria in diverse habitats can enable the design of media allowing their cultivation.

ResearchBlogging.orgBomar, L, et al. (2011). Directed culturing of microorganisms using metatranscriptomics. mBio, 2 (2) PMID: 21467263