FLEX(-) Bacterial DNA Extraction Protocol for Nanopore Sequencing v1

This protocol provides a rapid, high-throughput, and cost-effective method to extract high-quality, long-fragment genomic DNA from Gram-negative bacteria, optimized for producing DNA with sufficient purity, concentration, and fragment length for downstream molecular applications, with a primary focus on whole-genome sequencing using Oxford Nanopore R10 chemistry (including library preparation requirements). This workflow has been validated for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter spp., Stenotrophomonas maltophilia, and Serratia marcescens. In addition to Nanopore sequencing, the extracted DNA is suitable for common downstream workflows such as qPCR and other library preparation applications. The protocol can also be used for Illumina short-read sequencing; however, faster Illumina-specific extraction options may be available. In settings where both Nanopore and Illumina sequencing are performed in parallel, this protocol enables a single extraction to support both workflows, avoiding duplicate extraction steps. This method has been validated and implemented by the Department of Clinical Microbiology at Mount Sinai Hospital, and is currently used routinely to support sequencing workflows.