Increasing miRNA sequencing accuracy using an RNA circularization approach

Accurate quantification of all microRNAs (miRNA) is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. We have developed a new method of preparation of small RNA sequencing libraries (RealSeq®-AC) that ligates miRNAs with a single combo adapter and then circularizes the ligation products. When compared to other methods, RealSeq®-AC provided greatly reduced sequencing bias and allowed the identification of the largest variety of miRNAs in biological samples. The accuracy of detection of miRNAs was evaluated by sequencing miRNA-seq libraries in technical triplicates for each kit by using a synthetic pool of miRNAs (miRXplore pool, Miltenyi Biotec) and also a reference total RNA sample (Human Brain). The coverage of miRNA detection was evaluated by sequencing libraries prepared with TruSeq, RealSeq-AC, NEXTFlex, and QIAseq kits using a Universal miRNA Reference Kit (Agilent).