Abstract
The ability to accurately quantify all the microRNAs (miRNAs) in a sample is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. We develop a new method for preparing miRNA sequencing libraries, RealSeq®-AC, that involves ligating the miRNAs with a single adapter and circularizing the ligation products. When compared to other methods, RealSeq®-AC provides greatly reduced miRNA sequencing bias and allows the identification of the largest variety of miRNAs in biological samples. This reduced bias also allows robust quantification of miRNAs present in samples across a wide range of RNA input levels.
Highlights
MicroRNAs are small non-coding RNAs of approximately 21–23 nucleotides that control the expression of most genes and are involved in many biological processes
The major problem preventing the adoption of Next-generation sequencing (NGS) for routine expression profiling of miRNAs is its poor accuracy in quantifying many miRNA sequences: most individual miRNAs are underrepresented as a proportion of total sequence reads, in some cases by as much as 104-fold relative to their true abundance within a sample [3,4,5,6,7]
In our approach (Fig. 1b), we incorporate the sequences of both standard sequencing adapters into a single adapter. This single adapter is ligated to the 3′ end of the miRNA and the miRNA– adapter ligation product is circularized via intramolecular ligation
Summary
MicroRNAs (miRNAs) are small non-coding RNAs of approximately 21–23 nucleotides that control the expression of most genes and are involved in many biological processes. The major problem preventing the adoption of NGS for routine expression profiling of miRNAs is its poor accuracy (bias) in quantifying many miRNA sequences: most individual miRNAs are underrepresented as a proportion of total sequence reads, in some cases by as much as 104-fold relative to their true abundance within a sample [3,4,5,6,7]. In cases where sequencing bias leads to some miRNAs being under-detected or undetectable in particular samples, NGS can provide misleading or useless results [5, 7, 8]. It is likely that many important miRNAs remain to be discovered because they are not incorporated by current library preparation methods [9]
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