Current miRNA-Seq method yields inaccurate miRNA quantification. (A) Schematic of the two-step ligation protocol used to prepare small RNA libraries for deep sequencing. (B) Table of representative miRNAs from the 29 synthetic miRNA pool grouped by cluster where sequence differences are in red. (C) Representative result of deep sequencing from an equimolar mixture of the 29 synthetic miRNAs. Dashed line indicates expected cloning frequency for all, red bars indicate miRNAs with ≥50% GC content. Note the deviation is more than three orders of magnitude. (D) Deep sequencing data from previously published results, and one of our skin samples obtained from mouse hair follicle, grouped by miRNA clusters. Asterisks denote the absence of sequencing reads for a miRNA.