Pre-isolation Molecular Screening for High-throughput Sampling and Sequencing of Bacterial Microbes from the Environment

Environmental studies often require culture and characterization to understand the prevalence, distribution, persistence and functions of target microorganisms in ecological habitats. Isolating pure microbiological monocultures allows the phenotypic characterization of microorganisms to study their functional properties. For efficient isolation of low-prevalence organisms, enrichment followed by PCR screening is performed to identify positive samples for subsequent culture. Molecular characterization, strain-typing, and genotyping of isolated microorganisms is best comprehensively performed using whole-genome sequencing. This article outlines end-to-end protocols for screening, isolation, and sequencing of microbes from environmental samples. We provide systematic methods for environmental study design, enrichment, screening, and isolation of target microorganisms. Species identification is performed using qPCR or MALDI-TOF MS. Genomic DNA is extracted for whole-genome sequencing using the Oxford Nanopore platform. (c) 2023 Wiley Periodicals LLC.