Thursday, April 25, 2013

Introducing: TotalScript™ RNA-Seq Kit

RNA-seq without rRNA removal
The TotalScript Kit prepares high complexity RNA-Seq libraries from only 1-5 ng of intact total RNA, without the need for poly(A) enrichment or rRNA removal. Powered by Nextera™, TotalScript is index-capable, produces directional RNA-seq libraries, and is ideal for high quality RNA (RIN >7) from human, rodent, yeast, C. elegans and Drosophila. It is not recommended for prokaryotic samples.

The one-day procedure produces directional libraries, and is Index-capable (TotalScript™ Index kit sold separately). TotalScript can be adapted to desired rRNA content and transcript coverage based on chosen priming method.



Figure 1. Overview of the TotalScript™ RNA-Seq Kit process.

Friday, April 19, 2013

Co-evolution of an RNA virus and its fungal host

Among eukaryotes with modified nuclear genetic codes, viruses are unknown. This report provides data describing an RNA virus that infects a fungal host (Scheffersomyces segobiensis), with a derived nuclear genetic code where CUG codes for serine. To determine this information, Taylor et al. extracted total RNA from yeast cells using the MasterPure Yeast RNA Purification Kit. RNA-Seq was used to sequence the putative RNA virus, examine the tRNA expression of the host, test for host expression of known fungal viral sequences, and isolate host protein-coding sequences for bioinformatics analysis. Ribosomal RNA species were removed using the Ribo-Zero Magnetic Gold kit, and library was generated using ScriptSeq™ v 2 RNA-Seq Library Preparation Kit.  RNA-Seq was performed using 50-cycle paired-end runs on two flow cells of an Illumina® HiSeq 2000. A total of 409665 reads were mapped to the totivirus with an average coverage of 4404.80 times. The putative viral sequence was confirmed by Sanger sequencing using random and specific primers for cDNA library construction.

The genomic architecture and phylogeny are consistent with infection of the fungus by a double-stranded RNA virus of the genus Totivirus. The paper further provides evidence of past or present infection with totiviruses in five species of yeasts with modified genetic codes. All but one of the CUG codons in the viral genome have been eliminated, suggesting that avoidance of the modified codon was important to viral adaptation. Authors suggest that RNA viruses co-evolved with yeasts that underwent a major evolutionary transition from the standard genetic code.


ResearchBlogging.orgTaylor, D. (2013). Virus-host co-evolution under a modified nuclear genetic code PeerJ, 1 DOI: 10.7717/peerj.50