Anna Cusco from Vetgenomics in Spain spoke at the 2021 Nanopore Community Meeting about “Long-read metagenomics and Hi-C proximity ligation to characterize a canine fecal microbiome.” They have been characterizing microbiomes using metagenomics and Hi-C. The (now) classical approach is amplicon sequencing; however, it allows for taxonomic classification but not everything can be detected. With metagenomics, metagenome assembled genomes (MAGs) & long contigs can be produced. With metagenomics + HiC, the data allow for extra-chromosomal elements to be identified and long MAGs. The graphic that Cusco used compared amplicons, metagenomics, and metagenomics + Hi-C in terms of what can be identified. Cusco and team performed DNA extractions from a fecal sample of a healthy doc. They then split the sample for long-read metagenomics with the ONT ligation sequencing kit. The second part of the sample was used for Hi-C proximity ligation with the Proximeta Hi-C from Phase Genomics. High and medium-quality MAGs were used to analyze host and chromosomal elements. For the long-read metagenomics they used the MinION Mk1B, R9.4 flow cells, and the SQK-LSK 110 ligation sequencing kit. The sequences were basecalled with Guppy, corrected with Canu, assembled with Flye, and polished with Medaka. The Hi-C proximity ligation protocol used DNA cross-linking in vivo followed by cell lysis and fragmentation before proximity ligation and Illumina sequencing. Combining these approaches, they obtained 27 high-quality and 7 MAGs on canine feces. With long-read metagenomics only, 8 high-quality MAGs were obtained. MAGs recovered included those from closely related species and some considered novel. Five of the 27 MAGs seem to be canine-specific, explained Cusco. These canine-specific MAGs were from Allobaculum stercoricanis, for example. Long-read assemblies harboured more rRNA genes and were more contiguous compared to short-read assemblies. Canine MAGs contained prophages (from 0 to 3), and this was challenging for short-read data. Hi-C data allowed linking six plasmids to their bacterial hosts. Bacteriophages from CanMAGs clustered with gut phage database viruses. Cusco concluded that long-read metagenomics allowed retrieval of high-quality MAGs and long contigs harboring complete ribosomal operons, prophages, and mobile genetic elements. Hi-C allowed for linking of plasmids to hosts. The combination of Hi-C and long-read sequencing produced a more complete survey of the genomes and elements in Cooper (the dog’s) feces.
