Protocol for high-throughput processing of fecal samples for long-read metagenomic sequencing using PacBio HiFi or Oxford Nanopore Technologies

Long-read sequencing, whether using PacBio (PB) or Oxford Nanopore Technologies (ONT), requires high-molecular-weight (HMW) DNA at high purity and free of contaminants. Here, we present a protocol for high-throughput processing of fecal samples for long-read metagenomic sequencing. We describe steps for microbial inactivation, nucleic acid stabilization, and HMW DNA extraction. We then detail procedures for DNA cleanup, shearing, library preparation, and DNA sequencing. For complete details on the use and execution of this protocol, please refer to Minich et al.¹.