Small RNA Next Generation Sequencing without the bias

Selected Report:
https://www.newmarketscientific.com/datasheets/Randomized%20Adapters%20for%20Reducing%20Bias%20in%20Small%20RNA-Seq%20LibrariesV2.pdf

Small RNA NGS without the bias

SUMMARY

The past decade has seen an explosion of interest in the small RNA repertoires of animal and plant species, and in understanding the biological function of all types of small RNAs.

Next generation sequencing (NGS) is the most practical method for large-scale small RNA studies that aim to identify and enumerate small RNAs as with qPCR-based approaches, imperfectly matched small RNAs may still be able to hybridise to PCR primers. The same is also true for immobilised probes in microarrays. However NGS for small RNA analysis is also not without its challenges.

Small RNA libraries prepared for sRNA-Seq have been found to contain biases, resulting in libraries that inaccurately represent relative levels of the different small RNAs present in the starting RNA sample. This bias is caused by the T4-phage RNA ligases used during the ligation steps of small RNA library preparation. Ideally, the RNA ligases would show no preference for attaching adapters to small RNAs of different sequences, but in reality this is simply not the case.

A novel solution to overcome ligation bias in sRNA-seq libraries has been developed by Bioo Scientific (Austin TX USA), using a pool of adapters with randomised sequences at the ligation site, where each adapter sequence is present in vast molar excess over any given small RNA in the sample.

To demonstrate the reduced bias when using a randomised adapter strategy, small RNA sequencing libraries were prepared in triplicate from an equimolar mixture of 23 synthetic miRNAs, using either standard or randomised adapters in the ligation steps. Further details and the results are discussed in the link in this blog post.

Figure 1. Standard small RNA sequencing compared to NEXT flex Small RNA Sequencing Kit with randomised adapters.
Libraries were prepared from an equimolar mix of 23 synthetic miRNAs. Each slice in the pie graph represents one miRNA.

https://www.newmarketscientific.com/datasheets/Randomized%20Adapters%20for%20Reducing%20Bias%20in%20Small%20RNA-Seq%20LibrariesV2.pdf