TY - JOUR T1 - Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction JF - bioRxiv DO - 10.1101/474031 SP - 474031 AU - Hengyi Xu AU - Jun Yao AU - Douglas C. Wu AU - Alan M. Lambowitz Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/11/19/474031.abstract N2 - Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by (i) rationally designing RNA-seq adapters that minimize adapter dimer formation, and (ii) developing biochemical and computational methods that remediate 5’- and 3’-end biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5’- and 3’-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation. ER -