Statistical design approach enables optimised mechanical lysis for enhanced long-read soil metagenomics.

Metagenomic analysis has enabled insights into soil community structure and dynamics. Long-read sequencing for metagenomics can enhance microbial ecology by improving taxonomic classification, genome assembly, and functional annotation. However, protocols for purifying high-molecular weight DNA from soil are not yet optimised. We used a statistical design of experiments approach to enhance mechanical lysis of soil samples, increasing the length of purified DNA fragments. Low energy input into mechanical lysis improved DNA integrity, resulting in longer sequenced reads. Our optimized settings of 4 m s for 10 s increased fragment length by 70% compared to the manufacturer's recommendations. Longer reads from low intensity lysis produced longer contiguous sequences after assembly, potentially improving a range of down-stream analyses. Importantly, there was minimal bias exhibited in the microbial community composition due to lysis efficiency variations. We therefore propose a framework for improving the fragment lengths of DNA purified from diverse soil types, improving soil science research with long-read sequencing.