Soil nematode community profiling using reference-free mito-metagenomics

Nematodes are ubiquitous and diverse components of soil ecosystems worldwide. The 18S-based metabarcoding is known to have low species-level resolution and introduce bias in PCR. The mito-metagenomics (MMG) approach involves directly sequencing pooled samples, yields numerous mitochondrial reads that can be assembled into full or partial mitogenomes. This method circumvents the challenges associated with PCR-based metabarcoding and hold significant promise in biodiversity and phylogeny study. However, a reference database is typically required to extract mito-reads/contigs and provide taxonomic or phylogenetic context, thereby limiting its applicability. In this study, we introduced a novel reference-free pipeline for MMG assembly and diversity estimation. This pipeline has been integrated into a snakemake workflow, enabling the generation of output that is readily useable for phylogeny reconstruction in a single run. The performance tests have indicated that this new approach surpasses reference-based methods in soil nematode community profiling. We demonstrated that assembly quality improves with increasing sequencing depth, recommending an average of 1–2 Gb per species to achieve acceptable MMG assembly. Our pipeline presents an opportunity to create high-resolution phylogenies and assess diversity for poorly understood taxa, including neglected microscopic eukaryotes.