UC Irvine

High-throughput sequencing has become a standard and powerful tool for analyzing nucleic acids primarily due to its sensitivity and convenience. Small RNAs play important roles in regulating cellular and viral genes. The conventional methods for small RNA analyses are tedious and often lack accuracy, specificity and sensitivity for many small RNA species. Therefore, high-throughput sequencing becomes an indispensable tool for analyzing small RNAs. However, it is challenging to generate a reliable and representative small RNA library for high-throughput sequencing since small RNAs are usually expressed at extremely low levels and often contain modifications which affect library construction, usually causing biased readouts. This review compares various strategies for generating small RNA libraries of high quality and reliability, and provides recommendations on best practice in preparing high-throughput sequencing RNA libraries.