Evaluating bias-reducing protocols for RNA sequencing library preparation

Background

Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol).A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure.

Results

A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias.

Conclusions

The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-569) contains supplementary material, which is available to authorized users.     

Re-programming of translation following cell stress allows IRES-mediated translation to predominate

There is now an overwhelming body of evidence to suggest that internal ribosome entry is required to maintain the expression of specific proteins during patho-physiological situations when cap-dependent translation is compromised, for example, following heat shock or during mitosis, hypoxia, differentiation and apoptosis. Translational profiling has been used by several groups to assess the extent to which alternative mechanisms of translation initiation selectively recruit mRNAs to polysomes during cell stress. The data from these studies have shown that under each condition 3-5% of coding mRNAs remain associated with the polysomes. Importantly, the genes identified in each of these studies do not show a significant amount of overlap, suggesting that 10-15% of all mRNAs have the capability for their initiation to occur via alternative mechanism(s).     

A concomitant ATP-depleting strategy markedly enhances anticancer agent activity

Most anticancer agents effect DNA damage which initiate the cell death pathways of necrosis and apoptosis, but cancer cells of lesser sensitivity are only sublethally injured, and recover. The two death pathways and their interelationships in the presence of endogenous inhibitors of apoptosis and genetic deletions that facilitates only sublethal damage, are reviewed.Both ATP and pyrimidine levels in the sublethally injured cancer cells are reduced but not to low levels insuffient to sustain cell viability. However, this sublethal damage by the anticancer agent creates a therapeutic opportunity for further reduction of these key metabolites to lower levels that will not support life. Data in tumor-bearing animals is reviewed demonstrating that a combination of ATP-depleting agents plus a de novo pyrimidine inhibitor (PALA) administered concomitantly with each of nine different anticancer agents markedly enhances tumor regression rates, and even produces some cures. It is necessary to deplete tumor ATP levels seveerely (>85%) by a combination of agents that block both synthesis(6-methylmercaptopurine riboside, a purine de novo synthesis inhibitor) and generation of ATP(6-aminonicotin-amide, an inhibitor of glycolysis.) Cell viability cannot be sustained if the intracellular ATP level is reduced to 15% of normal or below. In vivo data employing this novel therapeutic strategy with cisplatin is presented. The potential significance of these findings to the improvement of cancer treatment is discussed.     

The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr

We have shown previously that polypyrimidine tract binding protein 1 (PTB) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr) is prebound. Here we show that the Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation.