Generating Whole Bacterial Genomes from Clinical Samples using a Target Enrichment Workflow.

Area of Science:

• Microbiology
• Genomics
• Infectious Diseases

Background:

• Obtaining complete bacterial genomes from clinical samples is difficult due to human DNA, microbiota, and low pathogen levels.
• Fastidious bacterial species and transport media hinder traditional culture-based methods for STI diagnosis.
• Direct genome sequencing often fails with low bacterial loads in clinical specimens.

Purpose of the Study:

• To develop and validate a target enrichment method for whole-genome sequencing of bacterial sexually transmitted infections (STIs) from clinical samples.
• To improve the success rate of genome recovery for STIs, especially those with low pathogen loads.
• To enable genomic surveillance and antimicrobial resistance profiling of bacterial STIs.

Main Methods:

• Utilized a probe panel targeting four bacterial STI species for whole-genome sequencing.
• Implemented target enrichment, a hybridization procedure, prior to genome sequencing.
• Optimized hybridization temperature to improve genome recovery for challenging samples, such as those with Mycoplasma genitalium.

Main Results:

• Achieved excellent genomic data from samples previously unsuitable for direct sequencing.
• Target enrichment demonstrated high effectiveness for bacterial STI genome sequencing, particularly with high pathogen loads (Ct < 30).
• Identified a new lineage of Chlamydia trachomatis lymphogranuloma venereum (ompA-genotype L4) in Buenos Aires.

Conclusions:

• Target enrichment is a highly effective method for sequencing bacterial STI genomes directly from clinical samples.
• This approach enhances genomic surveillance capabilities and aids in understanding antimicrobial resistance.
• The method facilitates the discovery of novel genomic diversity within bacterial STIs.