Oxford Nanopore Long-Read Sequencing of Bat Guano and Urine for Microbiome Characterization and Pathogen Detection

Deoxyribonucleic acid (DNA) sequencing has made possible the study of genetic sequences for taxonomic assignment, mutational analyses, and novel organism discovery. Long-read sequencing methods offer advantages over short-read methods because they can sequence entire DNA molecules without requiring fragmentation. This protocol describes a workflow for microbiome characterization and pathogen detection through the extraction, sequencing, and analysis of DNA from bat environmental samples, including guano and urine, using tailored approaches for each sample type. Pre-processing of guano and urine is performed prior to DNA extraction using a spin-column-based method to disrupt sample matrices and lyse microorganisms that may be present. This protocol further describes the preparation of sequencing libraries using either extracted DNA directly or polymerase chain reaction (PCR) products targeting the full-length 16S ribosomal RNA gene for prokaryotic and archaeal taxa and a partial 18S ribosomal RNA gene for eukaryotic taxa. PCR amplification of the full-length 16S ribosomal RNA gene is achieved using universal primers. Magnetic bead-based DNA purification is employed at multiple stages, including following PCR amplification of the 16S or 18S ribosomal RNA genes. Library preparation includes end repair, barcode ligation, pooling of barcoded DNA, and adapter ligation. Libraries are sequenced on a nanopore sequencing platform and analyzed using the EPI2ME bioinformatics workflow. Sequencing runs are performed on MinION flow cells, with run settings adjusted using the MinKNOW sequencing software. Runs are configured for 72 h with basecalling enabled for the raw data collected, generating the sequence-read files that are sorted according to barcode assignment. This workflow enables high-throughput sequencing of multiple samples in a single run, and the resulting sequence data are used for taxonomic classification of prokaryotic, archaeal, and eukaryotic organisms present in each sample, supporting wildlife microbiome characterization, environmental surveillance, and pathogen detection.