The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons. The core constituent of the small ribosomal subunit is a ~1.5 kb RNA strand in prokaryotes (16S rRNA) and a homologous ~1.8 kb RNA strand in eukaryotes (18S rRNA). Traditional sequencing-by-synthesis (SBS) of rRNA genes or rRNA cDNA copies has achieved wide use as a ‘molecular chronometer’ for phylogenetic studies, and as a tool for identifying infectious organisms in the clinic. However, epigenetic modifications on rRNA are erased by SBS methods. Here we describe direct MinION nanopore sequencing of individual, full-length 16S rRNA absent reverse transcription or amplification. As little as 5 picograms (~10 attomole) of purified E. coli 16S rRNA was detected in 4.5 micrograms of total human RNA. Nanopore ionic current traces that deviated from canonical patterns revealed conserved E . coli 16S rRNA 7-methylguanosine and pseudouridine modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E . coli strains.

Previously we showed that the protein unfoldase ClpX could facilitate translocation of individual proteins through the α-hemolysin nanopore. This results in ionic current fluctuations that correlate with unfolding and passage of intact protein strands through the pore lumen. It is plausible that this technology could be used to identify protein domains and structural modifications at the single-molecule level that arise from subtle changes in primary amino acid sequence (e.g., point mutations). As a test, we engineered proteins bearing well-characterized domains connected in series along an ∼700 amino acid strand. Point mutations in a titin immunoglobulin domain (titin I27) and point mutations, proteolytic cleavage, and rearrangement of beta-strands in green fluorescent protein (GFP), caused ionic current pattern changes for single strands predicted by bulk phase and force spectroscopy experiments. Among these variants, individual proteins could be classified at 86-99% accuracy using standard machine learning tools. We conclude that a ClpXP-nanopore device can discriminate among distinct protein domains, and that sequence-dependent variations within those domains are detectable.

The covalent modification of RNA molecules is a pervasive feature of all classes of RNAs and has fundamental roles in the regulation of several cellular processes. Mapping the location of RNA modifications transcriptome-wide is key to unveiling their role and dynamic behaviour, but technical limitations have often hampered these efforts. Nanopore direct RNA sequencing is a third-generation sequencing technology that allows the sequencing of native RNA molecules, thus providing a direct way to detect modifications at single-molecule resolution. Despite recent advances, the analysis of nanopore sequencing data for RNA modification detection is still a complex task that presents many challenges. Many works have addressed this task using different approaches, resulting in a large number of tools with different features and performances. Here we review the diverse approaches proposed so far and outline the principles underlying currently available algorithms.