Targeted nanopore sequencing for bacterial and viral classification

Webinar summary

Amplicon-based sequencing approaches have been widely adopted for routine microbiology applications, including targeted 16S rRNA for bacterial taxonomic classification and tiled multiplex PCR for viral whole-genome sequencing.
Our first speaker for this webinar, Dr. Joseph Petrone, discussed how to overcome the limitations of short-read methods in bacterial 16S rRNA sequencing, showcasing how to achieve deeper taxonomic resolution through nanopore sequencing of larger regions of rRNA gene operons. The pipeline, RESCUE, enables reproducible sequencing of bacterial rRNA operons, drastically improving species-level classification.Chris Kent then focused on viral whole-genome sequencing via tiling amplicon sequencing, emphasizing its cost-effectiveness and sensitivity. He discussed how novel algorithms and additional modes have been employed to enhance the versatility of tiling amplicon sequencing in microbiology research.

Learning objectives

• Resolve difficult-to-classify taxa present in microbial communities
• Address the limitations of short-read sequencing platforms
• Improve the performance of targeted microbiology approaches with nanopore technology

Abstract

Amplicon-based sequencing approaches have been widely adopted for routine microbiology applications, including targeted 16S rRNA for bacterial taxonomic classification and tiled multiplex PCR for viral whole-genome sequencing. In this webinar, you will hear from two speakers developing novel sequencing methods with Oxford Nanopore Technologies to improve the performance of targeted microbiology approaches over limitations associated with short-read sequencing technologies. First, Joseph Petrone demonstrated how, despite the advent of third-generation sequencing technologies, modern bacterial ecology studies still use traditional short-read sequencing technology to sequence small (~400bp) hypervariable regions of the 16S rRNA SSU to determine phylogenetic classification. By sequencing a larger region of the rRNA gene operons, the limitations and biases of sequencing small portions can be removed, allowing for more accurate classification with deeper taxonomic resolution. With nanopore sequencing now providing raw simplex reads with quality scores above Q20, the ease, cost, and portability of nanopore technology plays a leading role in performing differential bacterial abundance analysis. Sequencing the near-entire rrn operon of bacteria and archaea enables the use of the universally conserved operon holding evolutionary polymorphisms for taxonomic resolution. Here, a reproducible and validated pipeline was developed, RRN-operon Enabled Species-level Classification Using EMU (RESCUE), to facilitate sequencing of bacterial rrn operons and support import into phyloseq. The pipeline was validated through two complex mock samples, the use of multiple sample types, with actual traditional short-read sequencing data, and across four databases. RESCUE sequencing is shown to drastically improve classification to the species level for most taxa and resolves erroneous taxa caused by using traditional short-read sequencing technologies. Next, Christopher Kent discussed how tiling amplicon sequencing has emerged as a dominant method for viral whole-genome sequencing, due to its; low cost, high sensitivity, and accessibility. Furthermore, the experience gained from SARs-CoV-2 sequencing, makes this an ideal time to develop amplicon sequencing. However, amplicon sequencing has traditionally struggled with highly diverse viral clades, as variation within the primer binding sites affects the amplicon's efficacy. In this talk, examples of how these novel algorithms result in the generation of schemes with species-level variation. Furthermore, additional modes have been added; enabling the generation of typing panels, which produce amplicons targeting specific regions, across multiple pathogens.

Meet the speakers

Joseph Petrone

Joseph Petrone received his Ph.D. from the University of Florida’s Department of Microbiology and Cell Sciences under the mentorship of Dr. Eric Triplett. With a focus in bacterial genomics and microbiome analysis, some of his works involved comparative genomics of Diaphorina citri endosymbionts, high throughput rpsL mutation screening, and metagenomic analysis of the human microbiome. As his experience in bioinformatics and next-generation sequencing grew, Joseph began working towards developing a nanopore-based pipeline for 16S-ITS-23s rRNA analysis in order to achieve species-level classifications in samples that was previously out of reach. This led to publishing the RESCUE pipeline which is now being implemented across various human microbiome studies. Joseph’s interest in curating authentic genomic databases and developing new bioinformatic software led him to American Type Culture Collection (ATCC), where he now works as a bioinformatician for the Sequencing and Bioinformatics Center. His current project is now the development and curation of the ATCC Genome Portal.

Chris Kent

A PhD student working with Dr Joshua Quick and the ARTIC Network on furthering the capacity and applications of amplicon sequencing.