Abstract
Target enrichment is increasingly used for genotyping of plant and animal species or to better understand the evolutionary history of important lineages through the inference of statistically robust phylogenies. Limitations to routine target enrichment are both the complexity of current protocols and low input DNA quantity. Thus, working with tiny organisms such as microarthropods can be challenging. Here, we propose easy to set up optimizations for DNA extraction and library preparation prior to target enrichment. Prepared libraries were used to capture 1,432 ultraconserved elements (UCEs) from microhymenoptera (Chalcidoidea), which are among the tiniest insects on Earth and the most commercialized worldwide for biological control purposes. Results show no correlation between input DNA quantities (1.8-250 ng, 0.4ng with an extra whole genome amplification step) and the number of sequenced UCEs on an Illumina MiSeq. Phylogenetic inferences highlight the potential of UCEs to solve relationships within the families of chalcid wasps, which has not been achieved so far. The protocol (library preparation+target enrichment) allows processing 96 specimens in five working days, by a single person, without requiring the use of expensive robotic molecular biology platforms, which could help to generalize the use of target enrichment for minute specimens.
Highlights
Enriching subsets of the genome prior to sequencing allows effort to be concentrated on genomic regions that are relevant to answer specific research questions
Contributing to the global effort to solve the Hymenoptera tree of life while addressing the challenge of the phylogeny of chalcid wasps seemed sound. 115 Here, we provide a detailed description of the optimised protocol for DNA extraction and library preparation, followed by a description of the phylogenetic trees obtained through target enrichment of Ultra31 Conserved Elements (UCEs) from 96 species belonging to seven families and one subfamily of chalcids used for biological control (Aphelinidae, Azotidae, Encyrtidae, Eulophidae, Mymaridae, Pteromalidae: Eunotinae, Signiphoridae, Trichogrammatidae) as well as three
The number of UCEs obtained per individual appears to drop within the basal clades (i.e., Mymaridae and Trichogrammatidae, Figure S2), a result probably linked to the relatively long branches observed in these groups, and that could reduce the efficiency of the probes that were designed from the genome of Nasonia (Faircloth et al 2015)
Summary
Enriching subsets of the genome prior to sequencing (target enrichment, Mamanova et al 2010) allows effort to be concentrated on genomic regions that are relevant to answer specific research questions. Using this approach contributes to reducing cost, as experimental design can be adapted to sequence many samples simultaneously. Current protocols (DNA extraction, library preparation, target-enrichment) are time consuming and require handling expertise They have been initially developed to work with large amounts of input DNA (e.g., vertebrates or large/medium size insects; Faircloth et al 2015; McCormack et al 2013) and include many purification steps that increase DNA loss. Working on hyper[61] diverse groups of microarthropods is challenging, as it requires one to perform the extraction on i) a large number of specimens/species to be representative of the overall diversity of the group, without the possibility of using pipetting robots that increase DNA loss, ii) single individuals because species complexes are frequent (Al Khatib et al 2014; Kenyon et al 2015; Mottern & Heraty 2014), iii) the whole insect without destruction for vouchering and often prior to species identification, iv) rare species that have been collected once and may be represented in collection by a few specimens or only one specimen and, sometimes, v) old and dry museum specimens used for species description (types)
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