Evaluating bias-reducing protocols for RNA sequencing library preparation

Background Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol). A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure.