Poly-Adenylation Lengthening of Short RNAs for Simultaneous Short and Long Nanopore Sequencing (PALS-NS)

Sequencing of long coding RNAs informs about the abundance and the novelty in the transcriptome, while sequencing of short coding RNAs (e.g., microRNAs) or long non-coding RNAs informs about the epigenetic regulation of the transcriptome. Currently, each of these goals is addressed by separate sequencing experiments given the different physical characteristics of RNA species from biological samples. Sequencing of both short and long RNAs from the same experimental run has not been reported for long-read Nanopore sequencing to date and only recently has been achieved for short-read (Illumina) methods. We propose a library preparation method capable of simultaneously profiling short and long RNA reads in the same library on the Nanopore platform and provide the relevant bioinformatics workflows to support the goals of RNA quantification. Using a variety of synthetic samples we demonstrate that the proposed method can simultaneously detect short and long RNAs in a manner that is linear over 5 orders of magnitude for RNA abundance and three orders of magnitude for RNA length. In biological samples the proposed method is capable of profiling a wider variety of short and long non-coding RNAs when compared against the existing Smart-seq protocols for Illumina and Nanopore sequencing. Two by Two factorial design testing single tube polyadenylation reactions to extend the scope of long-read (Nanopore) sequencing to short (e.g. microRNA) and long (coding and non-coding) experiments