Cost-saving population genomic investigation of Daphnia longispina complex resting eggs using whole-genome amplification and pre-sequencing screening

Abstract

Resting stages of aquatic organisms that accumulate in the sediment over time are an exceptional resource that allows direct insights into past populations and addressing evolutionary questions. This is of particular interest in taxa that face relatively new environmental challenges, e.g., climate change and eutrophication, such as the Daphnia longispina species complex, a keystone zooplankton group in European freshwater ecosystems. However, genomic analysis might be challenging as DNA yield from many of these resting stages can be low and the material degraded. To reliably allow the resequencing of single Daphnia resting eggs from different sediment layers and characterize genomic changes through time, we performed whole-genome amplification to obtain DNA amounts suitable for genome resequencing and tested multiple protocols involving egg isolation, whole-genome amplification kits, and library preparation. A pre-sequencing contamination screening was developed, consisting of amplifying mitochondrial Daphnia and bacterial markers, to quickly assess and exclude possibly contaminated samples. In total, we successfully amplified and sequenced nine genomes from Daphnia resting eggs that could be identified as Daphnia longispina species. We analyzed the genome coverage and heterozygosity of these samples to optimize this method for future projects involving population genomic investigation of the resting egg bank.

Keywords: Daphnia longispina species complex; population genomics; resting egg bank; whole‐genome amplification.

FIGURE 1

Experimental design workflow for multiple protocols of whole‐genome amplification of isolated resting eggs, library preparation, sequencing, and bioinformatic analysis.

FIGURE 2

Proportion of reads mapped to a reference genome panel with FastQ Screen for all Illumina Nextera and NEB libraries. Reads that mapped to multiple genomes are not shown.

FIGURE 3

Normalized read depth across the genome in 10 kb bins for each sample. Scaffold positions are marked by alternating gray background bars. Coverage is shown on a log2 scale.

FIGURE 4

The proportion of heterozygous genotype calls in 250 kb sliding windows across the genome. Scaffold positions are marked by alternating gray background bars. Sample M5 is unamplified and the remaining eight are WGA samples using the same color code for the specific protocol as in Figure 3.