Small RNAs (sRNAs) are key regulators of genetic activity and accurate representation of sRNA in sequencing experiments is critical to the interpretation of biological data. NGS is now the gold standard for profiling sRNA and discovering new RNA. Further, NGS based approaches have now been extended to the study of protein function too. With such a heavy reliance on NGS, it is essential that the tools and protocols used in NGS generate accurate and reliable sequence data.
RNA ligases are essential in creating cDNA libraries prior to NGS sequencing. However, a number of recent publications reported that RNA ligases used in cDNA preparation actually mediate sequence specific ligation and the NGS approaches using these RNA ligases therefore do not represent all sRNA present in biological samples. The cause for sequence specific ligation is that the ligases preferentially ligate ends to each other that have more chance to be close to each other. This means that sRNAs that can efficiently anneal to the adapters have a higher chance to be ligated (Jayaprakash et al. 2011, Hafner et al. 2011 and Sorefan et al. 2012). These publications highlighted the limitations with the RNA ligases questioning the reliability of the currently widely used NGS approaches and the data generated from these approaches.
While identifying that cloning bias in sRNA libraries is RNA ligase dependent, Professor Tamas Dalmay, a renowned expert in siRNA biology from the School of Biological Sciences at the University of East Anglia, Norwich UK developed a novel, simple yet robust solution to overcome this problem (Sorefan et al. 2012). Dalmay and colleagues have developed a set of adapters that contain degenerated nucleotides therefore they are a pool of many sequences instead of one fixed sequence. Consequently, they give a chance to more different sRNAs to form a stable duplex with them leading to better coverage and more quantitative libraries. Professor Dalmay has shown that the novel tools and approach:
- doubles sequence coverage; compared to Illumina protocols there is an increase in sequencing coverage by up to 100%.
- gives more quantitative result that reflects better the true sRNA pool in the sample. Quantitative Northern blot confirmed the profile obtained by the new adapters, which was different from the profile obtained by Illumina adapters.
- Identifies previously un-cloned sequences. Using human cell lines they have shown that their protocol identifies more known miRNAs and crucially 11% more miRNAs (which were previously undiscovered).
The approach and tools developed by Dalmay have been evaluated by independent research groups who are convinced of the value of using this approach for obtaining reliable sequence data and true representation of micro RNA in biological samples.
The novel tools and approach developed by Professor Dalmay can make a genuine difference to NGS. The tools could be easily incorporated into existing sRNA cloning kits with
minimum costs and readily available for use. We envisage our novel approach could be applicable to SOLiD, although this is yet to be tested.
The novel tools and the approach are available for licensing by suitable industry partners to bring the product to market. University of East Anglia has a patent application (WO2013017861 A2) filed on this technology and we are now seeking for potential licensees, interested parties may contact Nathalie Horncastle at Research and Enterprise Services at the University of East Anglia, Norwich UK at N.Horncastle@uea.ac.uk (44)1603 591574.
- Hafner M, Renwick N, Brown M, Mihailović A, Holoch D, Lin C, Pena JTG, Nusbaum JD, Morozov P, Ludwig J, Ojo T, Luo S, Schroth G, Tuschl T: RNAligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA 2011, 17(9):1697–1712. [article]
- Jayaprakash AD, Jabado O, Brown BD, Sachidanandam R: Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res 2011, 39(21):e141. [article]
- Sorefan K, Griffiths-Jones S, Hall A , Kozomara A, Moulton V, Pais H, Dalmay T: Reducing ligation bias of small RNAs in libraries for next generation sequencing. Silence 2012, 3:4. [article]