Abstract
The expanding field of bacterial genomics has revolutionized our understanding of microbial diversity, biology and phylogeny. For most species, DNA extracted from culture material is used as the template for genome sequencing; however, the majority of microbes are actually uncultivable, and others, such as obligate intracellular bacteria, require laborious tissue culture to yield sufficient genomic material for sequencing. Chlamydiae are one such group of obligate intracellular microbes whose characterization has been hampered by this requirement. To circumvent these challenges, researchers have developed culture-independent sample preparation methods that can be applied to the sample directly or to genomic material extracted from the sample. These methods, which encompass both targeted [immunomagnetic separation-multiple displacement amplification (IMS-MDA) and sequence capture] and non-targeted approaches (host methylated DNA depletion-microbial DNA enrichment and cell-sorting-MDA), have been applied to a range of clinical and environmental samples to generate whole genomes of novel chlamydial species and strains. This review aims to provide an overview of the application, advantages and limitations of these targeted and non-targeted approaches in the chlamydial context. The methods discussed also have broad application to other obligate intracellular bacteria or clinical and environmental samples.
Highlights
From the cultivable minority to metagenomes to microbial genomesMicrobial community profiling and ecology analysis has proven to be a useful tool for discovering diverse, novel microbial taxa in the far reaches of our biosphere
While initial microbial diversity studies involved culture-dependent methods [1], the development and use of rRNA-based molecular methods [2, 3] led to the understanding that cultivable bacteria only represent ~1 % of the number of bacterial species in a given sample [4,5,6]
Microbial profiling tools quickly switched to culture-independent molecular methods, commonly using conserved regions of the 16S rRNA gene to characterize microbial diversity in environments such as soil, sediment and water, as well as human gut, skin and oral microbiomes [4,5,6,7,8,9]
Summary
From the cultivable minority to metagenomes to microbial genomesMicrobial community profiling and ecology analysis has proven to be a useful tool for discovering diverse, novel microbial taxa in the far reaches of our biosphere. There has been another shift toward deep metagenomic sequencing, in which the entire DNA extract is subject to shotgun sequencing [10,11,12], and single-cell genomics, whereby the genome of a single bacterium is sequenced exclusive of the background of its community [13,14,15,16,17,18] Both methods allow for the characterization of distinct microbial genomes. Microbial genomics is an everexpanding field, yet particular groups of bacteria are still challenging to sequence, such as obligate intracellular bacteria that require labour-intensive tissue culture and semipurification away from the host cells Helping to address these challenges are novel depletion or enrichment methods that target certain components of the sample [24,25,26,27,28], and are described in this review with reference to Chlamydiae
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