MG-MLST: Characterizing the Microbiome at the Strain Level in Metagenomic Data

Abstract

The microbiome plays an important role in human physiology. The composition of the human microbiome has been described at the phylum, class, genus, and species levels, however, it is largely unknown at the strain level. The importance of strain-level differences in microbial communities has been increasingly recognized in understanding disease associations. Current methods for identifying strain populations often require deep metagenomic sequencing and a comprehensive set of reference genomes. In this study, we developed a method, metagenomic multi-locus sequence typing (MG-MLST), to determine strain-level composition in a microbial community by combining high-throughput sequencing with multi-locus sequence typing (MLST). We used a commensal bacterium, Propionibacterium acnes, as an example to test the ability of MG-MLST in identifying the strain composition. Using simulated communities, MG-MLST accurately predicted the strain populations in all samples. We further validated the method using MLST gene amplicon libraries and metagenomic shotgun sequencing data of clinical skin samples. MG-MLST yielded consistent results of the strain composition to those obtained from nearly full-length 16S rRNA clone libraries and metagenomic shotgun sequencing analysis. When comparing strain-level differences between acne and healthy skin microbiomes, we demonstrated that strains of RT2/6 were highly associated with healthy skin, consistent with previous findings. In summary, MG-MLST provides a quantitative analysis of the strain populations in the microbiome with diversity and richness. It can be applied to microbiome studies to reveal strain-level differences between groups, which are critical in many microorganism-related diseases.

Comparative Genomic Analyses and CRISPR-Cas Characterization of Cutibacterium acnes Provide Insights Into Genetic Diversity and Typing Applications

“…Over the last decade, different approaches have been used to understand the genetic diversity and phylogenetic groups of C. acnes mainly based on single locus sequence analyses (16S rRNA, RecA , tly ) ( Scholz et al, 2014 ; McDowell, 2017 ; Yang et al, 2019 ), multilocus sequence typing (MLST) and more recently combined with whole genome sequence analyses ( Lomholt and Kilian, 2010 ; McDowell et al, 2011 , 2012 , 2013 ; Bangayan et al, 2020 ). While some of those typing methods can identify clonal complexes (CC) and sequence types (STs), they cannot differentiate among the sub-clades of type I strains.…”

“…As a result of the genetic diversity of Cutibacterium, recent efforts have focused on taxonomy and genotyping to compare strains associated with acne vulgaris. Originally, methods were based on PCR amplification and sequencing of variable sequences such as 16S rRNA, RecA, and tly, as well as multilocus sequence typing (MLST) encompassing seven to nine genes (McDowell et al, 2011(McDowell et al, , 2012(McDowell et al, , 2013Dreno et al, 2018;Bangayan et al, 2020). More recently, the advent of nextgeneration sequencing (NGS) and bioinformatic technologies has enabled the determination of the genomes of numerous isolates.…”

Genome-Based Targeted Sequencing as a Reproducible Microbial Community Profiling Assay

“…MA-GenTA bridges the gap between two commonly used approaches—16S rRNA gene sequencing and mWGS—combining some of the strengths of each ( Table 2 ). Other approaches have been described that attempt to balance cost with informativeness, including metagenomic multilocus sequence typing (MLST) ( 56 ), full-length 16S rRNA profiling ( 57 , 58 ), and shallow-coverage mWGS targeting as few as 0.5 million reads/sample ( 59 ).…”

Probiotics and live biotherapeutic products aiming at cancer mitigation and patient recover

“…The concept of healthy microbiomes playing an important role in human physiology has been built more recently in the history of Science, thanks to molecular biology techniques, mainly cloning and sequencing. Strain-level differences in microbiomes has allowed a better understanding of disease associations, not only with cancer, but with a host of diseases, and this have been achieved with a more detailed study in metagenomic sequencing and, therefore, access to complete genomes, as was the case with Propionibacterium acnes, being able to compare healthy skin microbiomes and those linked to acne, at strain level ( Bangayan et al, 2020 ). Studies using analytical chemistry showed the existence of a complex communication system among bacteria, signaled by quorum sensing molecules, a population density-dependent characteristic, and that can allow adaptations to environmental conditions, such those involving bioluminescence, antibiotic biosynthesis, plasmid conjugation and virulence ( Kiymaci et al, 2018 ).…”