Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications

Abstract

Nanopore direct RNA sequencing is a technology that allows the interrogation of the sequence information for epitranscriptomic modifications with the possibility of a quantitative assessment. The 113 pseudouridine (Ψ) sites...

Nanopore Direct RNA Sequencing for Modified Uridine Nucleotides Yields Signals Dependent on the Physical Properties of the Modified Base

“…[34,[44][45][46] We applied this approach to determine that stable bisulfite adducts to Ψ impact the ion current significantly such that the base call algorithm fails to provide a base call for the modification resulting in a deletion error for its presence in the RNA. [34] Further, this approach gives a positive signal change to minimize the false discovery rate of nanopore sequencing.…”

Bisulfite and Nanopore Sequencing for Pseudouridine in RNA

“…3 The pH 7 bisulfite reaction to yield Ψ adducts results in detection of adducts via a deletion signature in traditional sequencing after reverse transcription, 1,22,23 and it is found as an indel in nanopore direct RNA sequencing. 51 In the nanopore data, the indel results from the current levels being poorly recognized by the software. Future developments in nanopore data analysis software will allow this modified nucleotide and likely others to be directly sequenced.…”

“…coli. , The occupancy of Ψ across the transcriptome is organism, cell-type, and stress-dependent, suggesting that this U isomer has a significant role in cellular processes. ,,,, Determination of these roles will require accurate and quantitative sequencing of RNA, in which nanopore direct RNA sequencing is a candidate method to achieve this goal. , Our work identified that nanopore signatures for Ψ are highly sequence context dependent and that using a consensus of nanopore current, helicase dwell time, and base miscalling is an approach to sequence this U isomer . The pH 7 bisulfite reaction to yield Ψ adducts results in detection of adducts via a deletion signature in traditional sequencing after reverse transcription, ,, and it is found as an indel in nanopore direct RNA sequencing . In the nanopore data, the indel results from the current levels being poorly recognized by the software.…”

“…In our final study on this topic, we married the facile and clean pH 7 bisulfite reaction with Ψ to form a stable ribose adduct for nanopore direct RNA sequencing. 51 We reasoned that the size and charge of the adduct would dominate the signals in the nanopore and minimize or abolish the sequence dependency in the signatures. By producing a more coherent signal for Ψ via the adduct, quantification of these sites in nanopore data would be more easily achieved.…”

“…The pH 7 bisulfite reaction to yield Ψ adducts increases the (c) indel frequency at known modified sites and (d) shows no change in base calls for U/C sequence variations that give similar signals as Ψ in the nanopore data. Data to make this figure are from refs and .…”