Novel Method for Multiplex Small-RNA Library Preparation with Improved Performance and Higher Sensitivity

Identification and analysis of small RNA by deep sequencing requires preparation of a di-tagged cDNA library. Most library preparation methods for di-tagged cDNA are based on sequential ligation of adaptors. The excess of adaptors required to provide sufficient library yield leads to adaptor-dimer formation that strongly contaminates the library (unless several gel purification steps are performed to remove unligated adaptors). We have developed a novel method to generate di-tagged small RNA libraries free of adapter-dimer contamination without introducing any additional enzymatic steps or gel purifications. Using this simple and rapid procedure, library construction time is reduced to one day and the yield of the library is significantly improved by enabling the addition of a high concentration of adaptors, thereby increasing the percentage of small RNA molecules included in the library. To reduce sequencing cost and increase sample throughput we have developed a barcode strategy to tag samples during library construction. Up to 24 multiplexed libraries can then be pooled together before size selection, reducing the number of steps in the workflow. Additionally, gel-free size selection greatly simplifies the overall workflow. We have successfully used this method to generate libraries from human, mouse, rat, and plant RNA. Our method for generating multiplexed small RNA libraries reduces bias by ligation, increases representation of modified small RNAs and simplifies the workflow during library construction for small RNA analysis and discovery.