Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5 ' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs

Identification of full-length transcript isoforms using nanopore sequencing of individual RNA strands

Before RNA can be sequenced using next generation sequencing (NGS) technologies, it is first converted into cDNA (RNA-Seq). In 2016 Oxford Nanopore Technologies released their direct RNA nanopore sequencing technology, circumventing the requirement for cDNA. The native RNA is sequenced continuously from the 3' end through to the 5' end. Two limitations of this approach are: ambiguity in discriminating between full-length and truncated reads; and the requirement for a known invariable 3' end, such as the poly(A) tail. In collaboration with New England Biolabs, we developed a technique to identify full-length native RNA nanopore reads by specifically labeling capped RNA 5' ends with a nanopore detectable sequence. Using this strategy, we aimed to identify individual high-confidence full-length human mRNA isoform scaffolds among ~4 million nanopore poly(A)-selected RNA reads. First, we exchanged the biological 5' m$7G cap for a modified cap bearing a 45-nucleotide oligomer. This oligomer improved 5' end sequencing and ensured identification of capped strands. Second, among these capped reads, we screened for 3' ends consistent with documented polyadenylation sites. This gave 185,434 high-confidence mRNA scaffolds, including 4,262 that represented isoforms absent from GENCODE. Most of these had transcription start sites internal to longer, previously identified mRNA isoforms. Combined with orthogonal data, these mRNA scaffolds provide decisive evidence for full-length mRNA isoforms. In collaboration with the Ares lab, we developed a technique to label native RNA 3' ends with polyinosine. This step permits sequencing adapters to be ligated to both poly(A) and non-poly(A) RNA in a single sequencing experiment. Polyinosine tails are not known to naturally occur and produce a recognizable signal in nanopore ionic current data. These two features make it ideal for adapting a variety of RNA types while preserving native RNA 3' end sequence information. We implemented a Hidden Markov Model that identifies the polyinosine tail signal on the RNA 3' ends with 98.46% accuracy. This classifier can be used to filter the reads for a particular RNA 3' end type (e.g. separate nascent RNA from mature mRN

Identification of full-length transcript isoforms using nanopore sequencing of individual RNA strands

Before RNA can be sequenced using next generation sequencing (NGS) technologies, it is first converted into cDNA (RNA-Seq). In 2016 Oxford Nanopore Technologies released their direct RNA nanopore sequencing technology, circumventing the requirement for cDNA. The native RNA is sequenced continuously from the 3' end through to the 5' end. Two limitations of this approach are: ambiguity in discriminating between full-length and truncated reads; and the requirement for a known invariable 3' end, such as the poly(A) tail. In collaboration with New England Biolabs, we developed a technique to identify full-length native RNA nanopore reads by specifically labeling capped RNA 5' ends with a nanopore detectable sequence. Using this strategy, we aimed to identify individual high-confidence full-length human mRNA isoform scaffolds among ~4 million nanopore poly(A)-selected RNA reads. First, we exchanged the biological 5' m$7G cap for a modified cap bearing a 45-nucleotide oligomer. This oligomer improved 5' end sequencing and ensured identification of capped strands. Second, among these capped reads, we screened for 3' ends consistent with documented polyadenylation sites. This gave 185,434 high-confidence mRNA scaffolds, including 4,262 that represented isoforms absent from GENCODE. Most of these had transcription start sites internal to longer, previously identified mRNA isoforms. Combined with orthogonal data, these mRNA scaffolds provide decisive evidence for full-length mRNA isoforms. In collaboration with the Ares lab, we developed a technique to label native RNA 3' ends with polyinosine. This step permits sequencing adapters to be ligated to both poly(A) and non-poly(A) RNA in a single sequencing experiment. Polyinosine tails are not known to naturally occur and produce a recognizable signal in nanopore ionic current data. These two features make it ideal for adapting a variety of RNA types while preserving native RNA 3' end sequence information. We implemented a Hidden Markov Model that identifies the polyinosine tail signal on the RNA 3' ends with 98.46% accuracy. This classifier can be used to filter the reads for a particular RNA 3' end type (e.g. separate nascent RNA from mature mRN

Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing.

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

Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: application to direct RNA sequencing on nanopores

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3' nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3' ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3' ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3' end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores

Computational methods for RNA modification detection from nanopore direct RNA sequencing data

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.This paper was based upon work from COST Action CA16120 EPITRAN, supported by COST (European Cooperation in Science and Technology). AD-T is supported by an FPI Severo-Ochoa fellowship by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC). This work was partly supported by funds from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PGC2018-098152-A-100 to EMN), and by funds from the Italian Association for Cancer Research (AIRC, project IG 2020, ID. 24784 to MP). We acknowledge the support of the MEIC to the EMBL partnership, Centro de Excelencia Severo Ochoa and CERCA Programme/Generalitat de Cataluny

Discrimination among Protein Variants Using an Unfoldase-Coupled Nanopore

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 (<i>e.g.</i>, 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