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.

Chemical capping improves template switching and enhances sequencing of small RNAs.

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5' cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5' cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA-a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m7G capped ends. Second, among these 5'-capped nanopore reads, we screened for features consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m7G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.

Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other.

Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.

The yeast scavenger decapping enzyme DcpS and its application for in vitro RNA recapping.

Eukaryotic mRNAs are modified at their 5' end early during transcription by the addition of N7-methylguanosine (m7G), which forms the "cap" on the first 5' nucleotide. Identification of the 5' nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that the yeast scavenger decapping enzyme decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5' end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.