Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m⁶A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

Molecular biomarkers provide both diagnostic and prognostic results for patients with diffuse glioma, the most common primary brain tumor in adults. Here, we used a long-read nanopore-based sequencing technique to simultaneously assessIDHmutation status andMGMTmethylation level in 4 human cell lines and 8 fresh human brain tumor biopsies. Currently, these biomarkers are assayed separately, and results can take days to weeks. We demonstrated the use of nanopore Cas9-targeted sequencing (nCATS) to identifyIDH1andIDH2mutations within 36 h and compared this approach against currently used clinical methods. nCATS was also able to simultaneously provide high-resolution evaluation ofMGMTmethylation levels not only at the promoter region, as with currently used methods, but also at CpGs across the proximal promoter region, the entirety of exon 1, and a portion of intron 1. We compared the methylation levels of all CpGs toMGMTexpression in all cell lines and tumors and observed a positive correlation between intron 1 methylation andMGMTexpression. Finally, we identified single nucleotide variants in 3 target loci. This pilot study demonstrates the feasibility of using nCATS as a clinical tool for cancer precision medicine.

Streptococcus suisis an emerging zoonotic swine pathogen which can cause severe infections in humans. In March 2021, an outbreak ofS. suisinfections with 19 confirmed cases of septicemia and meningitis leading to two deaths, occurred in Nakhon Ratchasima province, Thailand. We characterized the outbreak through an epidemiological investigation combined with Illumina and Nanopore whole genome sequencing (WGS). The source of the outbreak was traced back to a raw pork dish prepared from a single pig during a Buddhist ceremony attended by 241 people. WGS analysis revealed that a singleS. suisserotype 2 strain belonging to a novel sequence type (ST) of the emergent Thai zoonotic clade CC233/379, was responsible for the infections. The outbreak clone grouped together with other Thai zoonotic strains from CC233/379 and CC104 in a globalS. suisphylogeny and capsule switching events between serotype 2 zoonotic strains and serotype 7 porcine strains were identified. The outbreak strain showed reduced susceptibility to penicillin corresponding with mutations in key residues in the penicillin binding proteins (PBPs). Furthermore, the outbreak strain was resistant to tetracycline, erythromycin, clindamycin, linezolid and chloramphenicol, having acquired an integrative and conjugative element (ICE) carrying resistance genestetOandermB,as well as a transposon from the IS1216 family carryingoptrAandermA. This investigation demonstrates that multi-drug resistant zoonotic lineages ofS. suiswhich pose a threat to human health continue to emerge.

With the advantages that long-read sequencing platforms such as Pacific Biosciences (Menlo Park, CA, USA) (PacBio) and Oxford Nanopore Technologies (Oxford, UK) (ONT) can offer, various research fields such as genomics and transcriptomics can exploit their benefits. Selecting an appropriate sequencing platform is undoubtedly crucial for the success of the research outcome, thus there is a need to compare these long-read sequencing platforms and evaluate them for specific research questions. This study aims to compare the performance of PacBio and ONT platforms for transcriptomic analysis by utilizing transcriptome data from three different tissues (hepatopancreas, intestine, and gonads) of the juvenile black tiger shrimp, Penaeus monodon. We compared three important features: (i) main characteristics of the sequencing libraries and their alignment with the reference genome, (ii) transcript assembly features and isoform identification, and (iii) correlation of the quantification of gene expression levels for both platforms. Our analyses suggest that read-length bias and differences in sequencing throughput are highly influential factors when using long reads in transcriptome studies. These comparisons can provide a guideline when designing a transcriptome study utilizing these two long-read sequencing technologies.