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

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 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 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 and bacterial markers, to quickly assess and exclude possibly contaminated samples. In total, we successfully amplified and sequenced nine genomes from resting eggs that could be identified as 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.