Optimal input DNA thresholds for genome skimming in marine crustacean zooplankton

Abstract

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.

Keywords: Copepoda; Euphausiacea; Genome skimming; Mitogenome; Zooplankton.

Figure 1. Workflow for genome skimming in this study.

Figure 2. Quality of sequence reads.

(A) Proportions of high-quality sequence reads after the quality-filtering step. (B) Proportions of sequence reads that remained if duplicated reads are removed (distinct reads). The numbers of reads used for analyses are listed in Table S2.

Figure 3. Mitochondrial genome sequences for different DNA inputs.

(A) Euphausia pacifica. (B) Calanus glacialis. Note that no >1,500 bp mitochondrial contigs were obtained for 10 and 1 pg samples of C. glacialis. Note that start and end points are different between reference sequences and assembled contigs.

Figure 4. The longest mitochondrial contig in different numbers of sequence reads for assembly.

The samples with 100 pg input DNA are used for Euphausia pacifica and Calanus glacialis.