Culture-independent approaches to chlamydial genomics

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.

Metagenomic Analysis of Fish-Associated Ca. Parilichlamydiaceae Reveals Striking Metabolic Similarities to the Terrestrial Chlamydiaceae

“…2013 ; Christiansen et al. 2014 ; Taylor-Brown et al. 2018 ), we opted for a nontargeted deep-sequencing approach, which provided insight into the chlamydial agent of interest against a background of gill microflora, and highlights an area of further study.…”

Whole-Genome Enrichment and Sequencing ofChlamydia trachomatisDirectly from Patient Clinical Vaginal and Rectal Swabs

“…Enrichment of the low copy number of C. trachomatis in clinical specimens presents the greatest challenge for culture-independent genome sequencing 48 . Initially, this methodology employed immunomagnetic separation (IMS) for enrichment, followed by genome amplification using multiple displacement amplification (MDA), but the demonstrated success rate (15-30%) was low across clinical specimens [49][50][51] .…”

“…Initially, this methodology employed immunomagnetic separation (IMS) for enrichment, followed by genome amplification using multiple displacement amplification (MDA), but the demonstrated success rate (15-30%) was low across clinical specimens [49][50][51] . Other methodologies currently in use include depletion-enrichment, cell-sorting-MDA, and multiplexed microdroplet PCR 48 . In 2014, Christiansen et al sequenced C. trachomatis from urine and vaginal swabs with an 80% (8/10) success rate (>95-100% coverage of the respective reference genome) using custom RNA baits to enrich for C. trachomatis during library preparation 52 .…”

“…In an effort to make direct sequencing of all C. trachomatis strains causing clinical infections more efficient, we designed an expanded RNA bait library (based on 85 C. trachomatis complete genomes, with 34,795, 120-mer probes instead of 74 genomes with 33,619, 120-mer probes as for previous bait libraries 48,52 ) and optimized the experimental protocol. The new system will increase our ability to sequence C. trachomatis genomes of current circulating strains causing ocular, urogenital and rectal infections in diverse populations and will provide a more robust data set to understand current sexual networks and transmission within and among anatomic sites.…”

Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria

“…Despite these successes, nanopore sequencing-based diagnostics still face the "needle in a haystack" problem of obtaining sufficient coverage of low-abundance target from a high-abundance background (e.g., pathogen/host, cancer/nontumor) sample 26 . While bacterial culture provides enriched quantities of genetic material in some applications 27 , culture-independent molecular biology-based target enrichment and background depletion methods 28 including amplification 29 and hybridization capture approaches 30 are increasingly being adapted for use in library preparation to yield "targeted" or "selective" sequencing 31,32 . Nearly all such methods require a priori knowledge to guide the design of the target-sequence-specific primers, baits, or probes required for selection.…”