Reducing Small RNA Sequencing Biases

from Genetic Engineering News by Adam R. Morris & Masoud M. Toloue

Randomized Adapter Strategy for Library Preparation Reduces Ligation Bias and Increases Accuracy of Small RNA-Seq

The study of small RNAs, including miRNAs, siRNAs, and pi-RNAs, is an ideal application of next-generation sequencing (NGS) technology. Although methods such as quantitative PCR and microarray analysis are useful for relative quantification of small RNAs, they suffer from two major drawbacks.

The first is that these methods are hybridization-based, which presents problems when trying to discriminate two small RNAs whose sequence differs by only a nucleotide or two. The second drawback is that both of these methods are only able to interrogate an a priori determined set of small RNAs, which both limits the scope of studies and prevents discovery of new small RNAs.

Both of these drawbacks of hybridization-based methods are addressed by using NGS for small RNA studies, as NGS can reliably distinguish small RNAs that differ by only a single base, and NGS is not limited to the study of a predetermined set of sequences. However, a major drawback of NGS methods for the study of small RNAs is the substantial bias that has been shown to exist in traditional library preparation protocols. This bias has been shown to be introduced during the two ligation steps, and the combined effect of the bias introduced in these steps results in some small RNAs being ligated to adapters much more efficiently than others.

Some of the studies that demonstrated the substantial bias introduced by RNA ligases showed that this bias resulted from the adapter sequence proximal to the ligation junction, and that adapters with 2–4 randomized bases at this junction could be used to substantially reduce ligation bias.

Jayaprakash et al. first demonstrated that NGS libraries prepared with this strategy showed little evidence of ligase bias and that data generated from these libraries correlated well with microarray and qPCR data.1 Bioo Scientific has obtained an exclusive license on this patent pending technology and has since developed a library preparation kit for high-throughput sequencing of small RNAs.

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Jayaprakash AD, et al. (2011) Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res 39(21): e141. [article]

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