Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections

Abstract

Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods.

Contribution of the patient microbiome to surgical site infection and antibiotic prophylaxis failure in spine surgery

“…In each case of SSI, targeted whole-genome enrichment of preoperative microbiome samples for the SSI strain was performed using a technique recently developed by our laboratory (GenCap-Seq; Fig. 1, F to K) ( 28 ). Interim analysis of the first 15 specimens using the standard GenCap-Seq protocol demonstrated limited and inconsistent enrichment, but specimens could be sufficiently interrogated using relatively large numbers of sequencing reads.…”

“…Alternative approaches using PCR amplicon sequencing of informative sites throughout the genome ( 40 ), synthetic capture-probe panels ( 41 ), or whole-genome amplification ( 42 ) have been applied in other contexts but carry limitations in terms of the genomic scope, scalability, bias, and need for organism-specific primer/probe optimizations, which may not be feasible because of the limited availability of clinical sample material. In this study, we used a targeted whole-genome enrichment technique recently developed by our laboratory (GenCap-Seq) ( 28 ) to implement a generalized, low-cost framework for the analysis of preoperative (metagenomic) and postoperative (SSI-isolate) pairs. Using this approach, we were able to examine lineage-specific polymorphisms in the preoperative microbiome at informative sites across the genome to assess the degree of similarity between preoperative strains and those subsequently isolated by clinical wound culture in cases of SSI.…”

“…For the most informative subset of patients who completed preoperative microbiome sampling and later developed SSI, we performed targeted enrichment of sequence reads from the SSI pathogen using GenCap-Seq ( 28 ). Briefly, metagenomic libraries were prepared using a Illumina DNA Prep with Enrichment kit ( 59 ).…”

“…The objectives of this prospective study were to characterize the contributions of the preoperative patient microbiome and resistome to SSI after instrumented spine surgery. Using multiple forms of genomic analysis, including techniques for species-targeted whole-genome enrichment of metagenomic sequencing libraries [genome capture sequencing (GenCap-Seq)] ( 28 ), we sought to determine (i) whether anatomic differences in the organisms causing SSI at various surgical anatomic locations ( 29 ) correlate with differences in the preoperative skin microbiome, (ii) whether the causative SSI strain(s) identified by clinical wound culture are present in the patient’s preoperative microbiota, and (iii) whether the preoperative resistome is related to the development of prophylaxis-resistant infection. In parallel, we also aimed to assess whether, under routine operating circumstances (in the absence of overt nosocomial outbreaks), SSIs occurring in a shared operative environment frequently involve cryptic transmission of exogenous, nosocomial strains across patients.…”

Co-inoculation with novel nodule-inhabiting bacteria reduces the benefits of legume-rhizobium symbiosis

“…These community members may be difficult to detect in amplicon sequencing data if their sequence counts in a sample are too low compared to other community members (Huse et al 2010; Paulson et al 2013; Escudié et al 2018). Given Sinorhizobium ’s extensive population growth and genome duplication within nodules (Mergaert et al 2006), sequencing technologies that enrich for particular targets, such as capture sequencing (Hayden et al 2022), may be required to detect low frequency non-rhizobial nodule occupants.…”