To make a short story long: simultaneous short and long RNA profiling on Nanopore devices

Abstract

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.

An Introduction to Nanopore Sequencing: Past, Present, and Future Considerations

“…Using a DNA adapter-ligation sequencing approach combined with modified bioinformatics, Jeck et al were able to successfully identify the BCR-ABL1 gene rearrangement (diagnostic hallmark for chronic myeloid leukemia [81]) and the PML-RAMA fusion within seconds of sequencing even with low library depth of the target fusions. While a common complaint of nanopore sequencing involves its high error rates, this group found that even low-quality base calls were mappable to the regions of interest, something that our group has also found [73]. Using similar methodology, the same group was later able to sequence these same libraries on ONT's Flongle device -their smallest, single-use flow cell that generates ~2.8 Gb of data with a cost below $100 -and were able to capture all of the previously-identified fusion genes and the fusion CIC-DUX4, which is embedded in a locus with a high number of repeats [82].…”

“…This makes them compatible for the adapter attachments that are necessary during library preparation and may also improve the polyadenylation-base enrichment methods [72]. Recent work by our group has demonstrated the ability to perform highly effective cosequencing of short and long coding and non-coding RNA species through universal poly-adenylation tailing, enabling contextualized quantification of all RNA species [73]. To further enhance the detection of target species, spike-in synthetic nucleotide mixes permit quantification, which we have demonstrated with mixed RNA samples using ERCC spike-in mixes [73], and others have demonstrated with RNA isoform identification and quantification using synthetic sequin RNAs [74].…”

“…Their method permitted discrimination of isoforms and methylation markers, though did not capture the full microRNA body. Interestingly this work suggests that the microRNA sequencing on ONT sequencers may be complicated by their short length, though subsequent investigation by our group has demonstrated the platform is capable of detecting even primer-dimer sized transcripts, and thus mi-croRNA sequences [73]. Circular RNA sequencing has also been performed on ONT sequencers, with protocols utilizing rolling-circle reverse transcription to capture full-length transcripts, enabling the detection of isoforms and fusion reads [98].…”

An Introduction to Nanopore Sequencing: Past, Present, and Future Considerations

“…Nanopore sequencing has been applied to these sample types because of its potential to deliver fusion gene readouts rapidly within 12 h. Using a DNA adapter-ligation sequencing approach combined with modified bioinformatics, Jeck et al were able to successfully identify the BCR-ABL1 gene rearrangement (a diagnostic hallmark for chronic myeloid leukemia [ 96 ]) and the PML-RAMA fusion within seconds of sequencing, even with low library depth of the target fusions. While a common complaint of nanopore sequencing involves its high error rate, this group found that even low-quality base calls were mappable to the regions of interest, something that our group has also found [ 89 ].…”

“…This makes them compatible for the adapter attachments that are necessary during library preparation, and may also improve the polyadenylation-base enrichment methods [ 88 ]. Recent work by our group has demonstrated the ability to perform highly effective cosequencing of short and long coding and noncoding RNA species through universal poly-adenylation tailing, enabling the contextualized quantification of all RNA species [ 89 ]. To further enhance the detection of target species, spike-in synthetic nucleotide mixes permit quantification, which we have demonstrated with mixed RNA samples using ERCC spike-in mixes [ 89 ], and others have demonstrated with RNA isoform identification and quantification using synthetic sequin RNAs [ 90 ].…”