Enrichment techniques for clinical metagenomics

Metagenomic next-generation sequencing (mNGS) offers a powerful, hypothesis-free approach for pathogen detection in clinical samples, allowing the identification of both known and novel microorganisms. However, the predominance of host nucleic acid in most samples poses a significant challenge, often overshadowing low-abundance pathogen sequences and increasing the cost of mNGS due to the high sequencing depth required. Enrichment techniques which selectively amplify pathogen-specific sequences can help to overcome this challenge, improving the sensitivity, specificity, and overall efficiency of mNGS - albeit while compromising the hypothesis-free nature and breadth of shotgun mNGS. As such, they can augment the use of mNGS in clinical scenarios where a more targeted approach is needed. This review provides a comprehensive analysis of the main enrichment techniques currently employed in the field, including PCR-based enrichment, CRISPR-Cas9 enrichment, molecular inversion probes (MIP), nanopore adaptive sequencing (AS), and hybridisation capture-based methods. We evaluate each method on a range of metrics including methodology, cost, sensitivity, specificity, and ease of integration into clinical workflows, as well as describing their application to date for purposes including pathogen detection, antimicrobial resistance profiling, and whole-genome sequencing across diverse clinical sample types. Current limitations and future directions for refinement and implementation of these techniques are also discussed. By summarising the current landscape and latest advancements in mNGS enrichment strategies, this review aims to guide the optimisation of mNGS workflows in clinical diagnostics and highlight key areas for future research.