Spatial Mapping of Mobile Genetic Elements and their Cognate Hosts in Complex Microbiomes - Combined MGE and taxonomic mapping

We used rRNA-FISH to stain five common oral genera, Veillonella, Streptococcus, Corynebacterium, Lautropia, and Neisseria, each with a different fluorophore, and we used MGE-FISH to stain the termL gene of the active prophage with a sixth fluorophore. We assembled contigs using combined long- and short-read sequencing and identified a highly abundant plasmid. Alignment of this contig to the plasmid database (PLSDB) showed that the plasmid had previously been observed in Prevotella nigrescens (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_018127865.1/). We selected two genes from the contig with metallo-β-lactamase (MBL) domains as targets for MGE-FISH (https://www.uniprot.org/uniprotkb/V8CNR4/entry, https://www.uniprot.org/uniprotkb/V8CNR9/entry). We stained both putative MBL genes (pMBL) with the same color using MGE-FISH. For taxonomic mapping, we broadened our target panel by employing HIPR-FISH. We selected a target panel of 18 genera that are highly abundant and prevalent in human plaque. We designed a HiPR-FISH spectral encoding using a 5-fluorophore combinatorial barcoding scheme, whereby each fluorophore represents a binary bit, providing 31 possible barcodes (2^5 - 1 = 31). The fluorophore for MGE-FISH was spectrally distinct from those of HiPR-FISH, enabling simultaneous implementation of both methods. Images are labeled by collection time such that the laser order for a given field of view (fov) is: 488nm Lambda, 514nm Lambda, 561nm Lambda, 633nm Airyscan, 405nm Lambda. We used OPERA-MS (https://github.com/CSB5/OPERA-MS) to do hybrid assembly with Illumina short reads and Nanopore long reads. The assemblies are in the fasta files and the reads that map to the assemblies are in the fastq files.