Strategies to study the metabolic origins of specialized plant metabolites: The specialized 1,4-naphthoquinones.

One of the hallmarks of specialized plant metabolites is that they are produced using precursors from central metabolism. Therefore, in addition to identifying and characterizing the pathway genes and enzymes involved in synthesizing a specialized compound, it is critical to study its metabolic origins. Identifying what primary metabolic pathways supply precursors to specialized metabolism and how primary metabolism has diversified to sustain fluxes to specialized metabolite pathways is imperative to optimizing synthetic biology strategies for producing high-value plant natural products in crops and microbial systems. Improved understanding of the metabolic origins of specialized plant metabolites has also revealed instances of recurrent evolution of the same compound, or nearly identical compounds, with similar ecological functions, thereby expanding knowledge about the factors driving the chemical diversity in the plant kingdom. In this chapter, we describe detailed methods for performing tracer studies, chemical inhibitor experiments, and reverse genetics. We use examples from investigations of the metabolic origins of specialized plant 1,4-naphthoquinones (1,4-NQs). The plant 1,4-NQs provide an excellent case study for illustrating the importance of investigating the metabolic origins of specialized metabolites. Over half a century of research by many groups has revealed that the pathways to synthesize plant 1,4-NQs are the result of multiple events of convergent evolution across several disparate plant lineages and that plant 1,4-NQ pathways are supported by extraordinary events of metabolic innovation and by various primary metabolic sources.

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.