NERD-seq: A novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs

Abstract

The new next-generation sequencing platforms by Oxford Nanopore Technologies for direct RNA sequencing (direct RNA-seq) allow for an in-depth and comprehensive study of the epitranscriptome by enabling direct base calling of RNA modifications. Non-coding RNAs constitute the most frequently documented targets for RNA modifications. However, the current standard direct RNA-seq approach is unable to detect many of these RNAs. Here we present NERD-seq, a sequencing approach which enables the detection of multiple classes of non-coding RNAs excluded by the current standard approach. Using total RNA from a tissue with high known transcriptional and non-coding RNA activity in mouse, the brain hippocampus, we show that, in addition to detecting polyadenylated coding and non-coding transcripts as the standard approach does, NERD-seq is able to significantly expand the representation for other classes of RNAs such as snoRNAs, snRNAs, scRNAs, srpRNAs, tRNAs, rRFs and non-coding RNAs originating from LINE L1 elements. Thus, NERD-seq presents a new comprehensive direct RNA-seq approach for the study of epitranscriptomes in brain tissues and beyond.

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing

“…Nanopore DRS is a well-established long-read sequencing technology to study RNA molecules, typically polyadenylated mRNAs [93][94][95][96][97] . Although several works have shown that this technology can also be used to study short RNA molecules, such as snoRNAs and snR-NAs 96,98 , DRS is inefficient at capturing RNA molecules shorter than 200 nucleotides (nt) and is generally considered unable to capture sequences shorter than ~100 nt 96,97 , limiting its applicability to study short RNA populations, such as tRNAs.…”

“…Nanopore DRS is a well-established long-read sequencing technology to study RNA molecules, typically polyadenylated mRNAs [93][94][95][96][97] . Although several works have shown that this technology can also be used to study short RNA molecules, such as snoRNAs and snR-NAs 96,98 , DRS is inefficient at capturing RNA molecules shorter than 200 nucleotides (nt) and is generally considered unable to capture sequences shorter than ~100 nt 96,97 , limiting its applicability to study short RNA populations, such as tRNAs. In addition, the first ~15 nt at the 5′ end of RNA molecules are typically lost in DRS runs 93,99 , as this portion cannot be adequately basecalled due to the increase in the RNA translocation speed when the 5′ end of the molecule exits the helicase 99 .…”