Novel tools for reducing bias in Next Generation Sequencing of small RNAs

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Published on: April 15, 2014

Tamas Dalmay, Professor of RNA Biology at the University of East Anglia (Norwich), has developed a robust, simple method of profiling small RNAs using next generation sequencing. Here he explains his novel HD adapters and why they are more reliable than existing commercial adapters. 

Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).
Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).

Small RNAs (sRNAs) are key regulators of gene expression, and accurate representation of sRNA in sequencing experiments is critical to the interpretation of biological data. Next generation sequencing (NGS) is now the gold standard for profiling and discovering new sRNAs, so it is essential that the tools and protocols used in NGS generate accurate, 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, so NGS approaches using these RNA ligases do not represent all sRNA present in biological samples. These publications highlighted the limitations associated with RNA ligases, questioning the reliability of currently widely used NGS approaches and the data generated from them.

Sequence specific ligation occurs because the ligases preferentially ligate ends that are more likely to be close to each other. This means that sRNAs that can efficiently anneal to the adapters have a higher chance of being ligated (Jayaprakash et al. 2011, Hafner et al. 2011 and Sorefan et al. 2012).

While identifying that cloning bias in sRNA libraries is RNA ligase dependent, our group at the School of Biological Sciences, University of East Anglia (Norwich), developed a novel, simple, robust solution to overcome this problem (Sorefan et al. 2012).

We developed a set of adapters (High Definition or HD adapters) that contain degenerated nucleotides, meaning they are a pool of many sequences instead of one fixed sequence. Consequently, many different sRNAs can form a stable duplex with them, leading to better coverage and more quantitative libraries. We have shown that using the HD adapters:

• Doubles sequence coverage – cDNA libraries generated through HD adapters contain about twice as many different sRNA sequences as libraries generated with standard Illumina adapter.

• Gives a more quantitative result that better reflects the true sRNA pool in the sample. We confirmed the profile obtained with our adaptors, which differed from the profile from Illumina adapters, by Northern blot.

• Identifies previously un-cloned sequences. Using human cell lines, we have shown that our protocol identifies more known miRNAs and crucially 11% more miRNAs (which were previously undiscovered).

Our approach and tools 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 microRNAs in biological samples.

The first publication describing the HD adapters was based on the Illumina sRNA cloning kit vs 1.5 (Sorefan et al., 2012), but we have now established a new protocol based on the TruSeq Illumina kit. The detailed protocol will be published soon but if anybody is interested in using it, please contact Tamas Dalmay ( We’re happy to send it to you ahead of publication.


• (OA) 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.

• (OA) 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.

• (OA) 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.


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