Abstract
Genomic analysis of hundreds of individuals is increasingly becoming standard in evolutionary and ecological research. Individual-based sequencing generates large amounts of valuable data from experimental and field studies, while using preserved samples is an invaluable resource for studying biodiversity in remote areas or across time. Yet, small-bodied individuals or specimens from collections are often of limited use for genomic analyses due to a lack of suitable extraction and library preparation protocols for preserved or small amounts of tissues. Currently, high-throughput sequencing in zooplankton is mostly restricted to clonal species, that can be maintained in live cultures to obtain sufficient amounts of tissue, or relies on a whole-genome amplification step that comes with several biases and high costs. Here, we present a workflow for high-throughput sequencing of single small individuals omitting the need for prior whole-genome amplification or live cultures. We establish and demonstrate this method using 27 species of the genus Daphnia, aquatic keystone organisms, and validate it with small-bodied ostracods. Our workflow is applicable to both live and preserved samples at low costs per sample. We first show that a silica-column based DNA extraction method resulted in the highest DNA yields for nonpreserved samples while a precipitation-based technique gave the highest yield for ethanol-preserved samples and provided the longest DNA fragments. We then successfully performed short-read whole genome sequencing from single Daphnia specimens and ostracods. Moreover, we assembled a draft reference genome from a single Daphnia individual (>50× coverage) highlighting the value of the workflow for non-model organisms.
Highlights
Small-bodied species are interesting for ecological and evolutionary research as they have the potential to rapidly adapt to environmental change due to higher levels of genetic variation (Ellegren & Galtier, 2016), shorter generation times (Blueweiss et al, 1978), and potentially faster molecular evolution (Martin & Palumbi, 1993; Thomas, Welch, Lanfear, & Bromham, 2010)
Using single individuals instead of pooled samples improves estimates of allele frequencies (Dorant et al, 2019), aids the identification of genes associated with environmental variation (Rellstab, Gugerli, Eckert, Hancock, & Holderegger, 2015) or phenotypes (Kratochwil, Urban, & Meyer, 2019), and the identification of population structure (Ekblom & Wolf, 2014)
Preserved samples from archives and collections stored in museums, institutes, or universities, offer vast opportunities for phylogenomic analyses (Evans et al, 2019) or to study temporal changes
Summary
Small-bodied species are interesting for ecological and evolutionary research as they have the potential to rapidly adapt to environmental change due to higher levels of genetic variation (Ellegren & Galtier, 2016), shorter generation times (Blueweiss et al, 1978), and potentially faster molecular evolution (Martin & Palumbi, 1993; Thomas, Welch, Lanfear, & Bromham, 2010). High-throughput sequencing of small organisms has so far been performed by including an WGA step (Cruaud et al, 2019; Grealy, Bunce, & Holleley, 2019; Lack, Weider, & Jeyasingh, 2017) This technique enables the use of small amounts of DNA, but introduces biases due to PCR selection, PCR artefacts, and PCR drift (Sabina & Leamon, 2015). It is paramount that techniques of DNA extraction and library preparation are improved to leverage the power of high-throughput sequencing techniques for genomic studies with small-bodied and preserved individuals. We combined all these approaches into a workflow (Figure 1) detailing the steps from sample to high-quality sequencing result
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