PoLoCo: A reproducible pooled low-coverage workflow for draft genome assembly and allele frequency analysis from ethanol-preserved small non-model invertebrates

Abstract Background Many ecologically important small invertebrates are preserved in ethanol, which compromises DNA integrity and severely limits their potential to be utilized in further genomic research. The lack of soil arthropod representation ultimately results in limited availability of reference genomes and population genomic inference from pooled sequencing (Pool-seq). Results We introduce PoLoCo (Pooled Low-Coverage), a reproducible workflow integrating pooled specimen draft genome assembly from ethanol-preserved specimens with population-level allele-frequency analysis. We assembled a 411 Mb draft genome of Entomobrya nivalis from pooled short reads, achieving 97.7% BUSCO completeness despite notable fragmentation. Comparison with the published reference genome showed partial genomic correspondence while also revealing reference-dependent differences in the retained SNP datasets. The application of PoLoCo to 82 retained pooled populations produced a final distance-thinned allele-frequency dataset suitable for population-scale genomic inference. Case-study execution documented the computational requirements of the major workflow components on standard HPC infrastructure, with the workflow, documentation, and software environments openly available in Zenodo. Conclusions PoLoCo demonstrates that draft genome assemblies and reproducible Pool-seq allele-frequency analyses can be performed with long-preserved sample material from the field. PoLoCo integrates reference construction, sync-based allele-frequency analysis, and benchmarking into a free and open workflow. It establishes a reproducibility benchmark for biodiversity genomics and yields a transferable workflow for ecological and evolutionary initiatives where high-quality DNA or long-read sequencing is not feasible.