Long-Read Metagenomics and CAZyme Discovery.

Microorganisms play a primary role in regulating biogeochemical cycles and are a valuable source of enzymes that have biotechnological applications, such as carbohydrate-active enzymes (CAZymes). However, the inability to culture the majority of microorganisms that exist in natural ecosystems restricts access to potentially novel bacteria and beneficial CAZymes. While commonplace molecular-based culture-independent methods such as metagenomics enable researchers to study microbial communities directly from environmental samples, recent progress in long-read sequencing technologies are advancing the field. We outline key methodological stages that are required as well as describe specific protocols that are currently used for long-read metagenomic projects dedicated to CAZyme discovery.

Mobilome-driven segregation of the resistome in biological wastewater treatment.

Biological wastewater treatment plants (BWWTP) are considered to be hotspots for the evolution and subsequent spread of antimicrobial resistance (AMR). Mobile genetic elements (MGEs) promote the mobilization and dissemination of antimicrobial resistance genes (ARGs) and are thereby critical mediators of AMR within the BWWTP microbial community. At present, it is unclear whether specific AMR categories are differentially disseminated via bacteriophages (phages) or plasmids. To understand the segregation of AMR in relation to MGEs, we analyzed meta-omic (metagenomic, metatranscriptomic and metaproteomic) data systematically collected over 1.5 years from a BWWTP. Our results showed a core group of 15 AMR categories which were found across all timepoints. Some of these AMR categories were disseminated exclusively (bacitracin) or primarily (aminoglycoside, MLS and sulfonamide) via plasmids or phages (fosfomycin and peptide), whereas others were disseminated equally by both. Combined and timepoint-specific analyses of gene, transcript and protein abundances further demonstrated that aminoglycoside, bacitracin and sulfonamide resistance genes were expressed more by plasmids, in contrast to fosfomycin and peptide AMR expression by phages, thereby validating our genomic findings. In the analyzed communities, the dominant taxon Candidatus Microthrix parvicella was a major contributor to several AMR categories whereby its plasmids primarily mediated aminoglycoside resistance. Importantly, we also found AMR associated with ESKAPEE pathogens within the BWWTP, and here MGEs also contributed differentially to the dissemination of the corresponding ARGs. Collectively our findings pave the way toward understanding the segmentation of AMR within MGEs, thereby shedding new light on resistome populations and their mediators, essential elements that are of immediate relevance to human health.

Functional meta-omics provide critical insights into long- and short-read assemblies.

Real-world evaluations of metagenomic reconstructions are challenged by distinguishing reconstruction artifacts from genes and proteins present in situ. Here, we evaluate short-read-only, long-read-only and hybrid assembly approaches on four different metagenomic samples of varying complexity. We demonstrate how different assembly approaches affect gene and protein inference, which is particularly relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic and metaproteomic data to assess the metagenomic data-based protein predictions. Our findings pave the way for critical assessments of metagenomic reconstructions. We propose a reference-independent solution, which exploits the synergistic effects of multi-omic data integration for the in situ study of microbiomes using long-read sequencing data.