Optimal input DNA thresholds for genome skimming in marine crustacean zooplankton

Crustacean zooplanktons are key secondary and tertiary producers in marine ecosystems, yet their genomic resources remain poorly understood. To advance biodiversity research on crustacean zooplankton, this study evaluated the effectiveness of genome skimming, a method that assembles genetic regions, including mitogenome, from shotgun genome sequencing data. Because the small amount of DNA available is a limitation in zooplankton genetics, different input DNA amounts (1 pg-10 ng) were prepared for library construction for genome skimming using two large species: Euphausia pacifica (Euphausiacea) and Calanus glacialis (Copepoda). Additionally, de novo assembly was used to obtain long contigs from short reads because reference-guided assembly can not be applied to all crustacean zooplankton. Evaluation of the raw sequence reads showed increased proportions of high-quality and distinct reads (low duplication levels) for large DNA inputs. By contrast, low sequence quality and high sequence duplication were observed for ≤ 10 pg DNA samples, owing to increased DNA amplification cycles. Complete mitogenomes, including all 37 genes, were successfully retrieved for ≥ 10 pg (E. pacifica) and ≥ 100 pg (C. glacialis) of DNA. Despite the large estimated genome sizes of these zooplankton species, only ≥ 1 and ≥ 3 M reads were sufficient for mitogenome assembly for E. pacifica and C. glacialis, respectively. Nuclear ribosomal repeats and histone 3 were identified in the assembled contigs. As obtaining sufficient DNA amounts (≥ 100 pg) is feasible even from small crustacean zooplankton, genome skimming is a powerful approach for robust phylogenetics and population genetics in marine zooplankton.