Global mpox lineage discovery and rapid outbreak tracking with nanopore sequencing.

Insufficient tracking of virus introduction, spread, and new lineage emergence for the human monkeypox (mpox) virus 1 (hMPXV1) outbreak of 2022 hindered epidemiological studies and public health response. hMPXV1 mutations accumulated unexpectedly faster than predicted. Thus, new variants with altered pathogenicity could emerge and spread without early detection. Whole genome sequencing addresses this gap when implemented but requires widely accessible and standardized methodologies to be effective both regionally and globally. Here we developed a rapid nanopore whole genome sequencing method complete with working protocols, from DNA extraction to phylogenetic analysis tools. Using this method, we sequenced 84 complete hMPXV1 genomes from Illinois, a Midwestern region of the United States, spanning the first few months of the outbreak. The resulting five-fold increase in hMPXV1 genomes from this region established two previously unnamed global lineages, several mutational profiles not seen elsewhere, multiple separate introductions of the virus into the region, and the likely emergence and spread of new lineages from within this region. These results demonstrate that a dearth of genomic sequencing of hMPXV1 slowed our understanding and response to the mpox outbreak. This accessible nanopore sequencing approach makes near real-time mpox tracking and rapid lineage discovery straightforward and creates a blueprint for how to deploy nanopore sequencing for genomic surveillance of diverse viruses and future outbreaks.

Efficient Cloning and Sequence Validation of Repetitive and High GC-Content Short Hairpin RNAs.

Short hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.1 vectors. Improvements included shRNA insert design and preparation, recombination-based cloning, and sequencing-based validation that included Sanger and nanopore long-read sequencing. This improved method should enable practical, efficient cloning of nearly any shRNA sequence.

High throughput nanopore sequencing of SARS-CoV-2 viral genomes from patient samples.

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable "how-to" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.