LITERATURE UPDATE


remains challenging. In this study, the authors developed a sequence-based functional metagenomics procedure for mining the diversity of copper (Cu) resistance gene copA in global microbiomes, by combining the metagenomic assembly technology, local BLAST, evolutionary trace analysis (ETA), chemical synthesis, and conventional functional genomics.


In total, 87 metagenomes were collected from a public database and subjected to copA detection, resulting in 93,899 hits. Manual curation of 1214 hits of high confidence led to the retrieval of 517 unique CopA candidates, which were further subjected to ETA. Eventually, 175 novel copA sequences of high quality were discovered. Phylogenetic analysis showed that almost all these putative CopA proteins were distantly related to known CopA proteins, with 55 sequences from totally unknown species. Ten novel and three known copA genes were chemically synthesised for further functional genomic tests using the Cu-sensitive Escherichia coli (ΔcopA). The growth test and Cu uptake determination showed that five novel clones had positive effects on host Cu resistance and uptake. One recombinant harbouring copA-like 15 (copAL15) successfully restored Cu resistance of the host with a substantially enhanced Cu uptake. Two novel copA genes were fused with the gfp gene and expressed in E. coli for microscopic observation. Imaging results showed that they were successfully expressed and their proteins were localised to the membrane. The results presented here greatly expand the diversity of known CopA proteins, and the sequence-based procedure developed overcomes biases in length, screening methods, and abundance of conventional functional metagenomics.


Hecatomb: an integrated software platform for viral metagenomics Roach MJ, Beecroft SJ, Mihindukulasuriya KA et al. Gigascience. 2024 Jan 2;13:giae020. doi: 10.1093/gigascience/giae020.


Modern sequencing technologies offer extraordinary opportunities for virus discovery and virome analysis. Annotation of viral sequences from metagenomic data requires a complex series of steps to ensure accurate annotation of individual reads and assembled contigs. In addition, varying study designs will require project-specific statistical analyses. Here, the authors introduce


Hecatomb, a bioinformatic platform coordinating commonly used tasks required for virome analysis. Hecatomb means “a great sacrifice.” In this setting, Hecatomb is “sacrificing” false-positive viral annotations using extensive quality control and tiered-database searches. Hecatomb processes metagenomic data obtained from both short- and long-read sequencing technologies, providing annotations to individual sequences and assembled contigs. Results are provided in commonly used data formats useful for downstream analysis. Here, the authors demonstrate the functionality of Hecatomb through the reanalysis of a primate enteric and a novel coral reef virome.


Hecatomb provides an integrated


platform to manage many commonly used steps for virome characterisation, including rigorous quality control, host removal, and both read- and contig- based analysis. Each step is managed using the Snakemake workflow manager with dependency management using Conda. Hecatomb outputs several tables properly formatted for immediate use within popular data analysis and visualisation tools, enabling effective data interpretation for a variety of study designs. Hecatomb is hosted on GitHub (github.com/shandley/hecatomb) and is available for installation from Bioconda and PyPI.


Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction


Simon SA, Schmidt K, Griesdorn L, Soares AR, Bornemann TLV, Probst AJ. BMC Genomics. 2023 Dec 1;24(1):727. doi: 10.1186/s12864-023-09853-w.


While genome-resolved metagenomics has revolutionised our understanding of microbial and genetic diversity in environmental samples, assemblies of short-reads often result in incomplete and/or highly fragmented metagenome- assembled genomes (MAGs), hampering in-depth genomics. Although Nanopore sequencing has increasingly been used in microbial metagenomics as long reads greatly improve the assembly quality of MAGs, the recommended DNA quantity usually exceeds the recoverable amount of DNA of environmental samples. Here, the authors evaluated lower- than-recommended DNA quantities for Nanopore library preparation by determining sequencing quality, community composition, assembly quality and recovery of MAGs. The authors generated 27 Nanopore metagenomes using the commercially


WWW.PATHOLOGYINPRACTICE.COM JUNE 2025


available ZYMO mock community and varied the amount of input DNA from 1000 ng (the recommended minimum) down to 1 ng in eight steps. The quality of the generated reads remained stable across all input levels. The read mapping accuracy, which reflects how well the reads match a known reference genome, was consistently high across all libraries. The relative abundance of the species in the metagenomes was stable down to input levels of 50 ng. High-quality MAGs (>95% completeness, ≤5% contamination) could be recovered from metagenomes down to 35 ng of input material. When combined with publicly available Illumina reads for the mock community, Nanopore reads from input quantities as low as 1 ng improved the quality of hybrid assemblies. The results show that the


recommended DNA amount for Nanopore library preparation can be substantially reduced without any adverse effects to genome recovery and still bolster hybrid assemblies when combined with short-read data. The authors posit that the results presented herein will enable studies to improve genome recovery from low-biomass environments, enhancing microbiome understanding.


Mycobiome and Mycobiome- Associated Diseases Hagihara M, Kato H, Shibata Y et al. Med Mycol J. 2023;64(3):55-62. doi: 10.3314/mmj.23-002.


The human body is host to a large number of commensal microbial species such as bacteria, fungi and viruses. Among these, the human mycobiome is often neglected as a potential cause of disease, as it is thought to be comparatively much less abundant and less diverse than the human bacteriome. Additionally, most fungi are not easily cultured, even in specific media. Hence, their study has been limited to date, mainly because of the unavailability of methods used for their detection. However, the utilisation of a novel metagenomic methodology will enable the identification of well- characterised mycobiomes in several parts of the human body and broaden our knowledge of their contribution to human health and disease. In this article, the authors review the


role of the human mycobiome in the gut, respiratory organs, skin, genital tract, and carcinogenesis, highlighting the correlations between the human mycobiome and mycobiome-associated diseases.


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