bioRxiv preprint doi: https://doi.org/10.1101/437590; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Optimised DNA extraction and library preparation for minute arthropods: application 2 to target enrichment in chalcid wasps used for biocontrol. 3 4 Astrid Cruaud1*, Sabine Nidelet1, Pierre Arnal1, 2, Audrey Weber3, Lucian Fusu4, Alex 5 Gumovsky5, John Huber6, Andrew Polaszek7 and Jean-Yves Rasplus1 6 7 1 CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France 8 2 ISYEB–UMR 7205 MNHN, CNRS, UPMC, EPHE, Sorbonne Universités, Paris, France 9 3 AGAP, INRA, CIRAD, Montpellier SupAgro, Univ Montpellier, Montpellier, France. 10 4 Faculty of Biology, Alexandru Ioan Cuza University, Iasi, Romania 11 5 Schmalhausen Institute of Zoology, National Academy of Sciences of Ukraine, Kiev, 12 Ukraine 13 6 Natural Resources Canada, c/o Canadian National Collection of Insects, Ottawa, Canada 14 7 Department of Life Sciences, Natural History Museum, London, UK 15 16 *corresponding author: [email protected] 17 18 running headline = target-enrichment in minute wasps 19 20 Abstract 21 Enriching subsets of the genome prior to sequencing allows focusing effort on regions that are 22 relevant to answer specific questions. As experimental design can be adapted to sequence 23 many samples simultaneously, using such approach also contributes to reduce cost. In the 24 field of ecology and evolution, target enrichment is increasingly used for genotyping of plant 25 and animal species or to better understand the evolutionary history of important lineages 1 bioRxiv preprint doi: https://doi.org/10.1101/437590; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 26 through the inference of statistically robust phylogenies. Limitations to routine target 27 enrichment by research laboratories are both the complexity of current protocols and low 28 input DNA quantity. Thus, working with tiny organisms such as micro-arthropods can be 29 challenging. Here, we propose easy to set up optimisations for DNA extraction and library 30 preparation prior to target enrichment. Prepared libraries were used to capture 1432 Ultra- 31 Conserved Elements (UCEs) from microhymenoptera (Chalcidoidea), which are among the 32 tiniest insects on Earth and the most commercialized worldwide for biological control 33 purposes. Results show no correlation between input DNA quantities (1.8-250ng, 0.4 ng with 34 an extra whole genome amplification step) and the number of sequenced UCEs. Phylogenetic 35 inferences highlight the potential of UCEs to solve relationships within the families of chalcid 36 wasps, which has not been achieved so far. The protocol (library preparation + target 37 enrichment), allows processing 96 specimens in five working days, by a single person, 38 without requiring the use of expensive robotic molecular biology platforms, which could help 39 to generalize the use of target enrichment for minute specimens. 40 41 42 Key words : Chalcidoidea, library construction, low DNA quantity, micro-arthropods, target 43 enrichment, UCEs. 44 2 bioRxiv preprint doi: https://doi.org/10.1101/437590; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 45 INTRODUCTION 46 Enriching subsets of the genome prior to sequencing (target enrichment, Mamanova et al. 47 2010) allows effort to be concentrated on genomic regions that are relevant to answer specific 48 research questions. Using this approach also contributes to reducing cost, as experimental 49 design can be adapted to sequence many samples simultaneously. In the fields of ecology and 50 evolution, target enrichment has been used for genotyping or phylogenomics of plant and 51 animal species (Gasc et al. 2016; Lemmon & Lemmon 2013), to characterize phenotypic 52 traits (e.g., Muraya et al. 2015) or to explore microbial ecosystems (Gasc & Peyret 2018). 53 54 However, routine target enrichment by research laboratories is limited both by the complexity 55 of current protocols, and by input DNA quantity that may be very low for some minute 56 species (e.g. micro-arthropods < 1mm) and /or old /rare (museum) specimens. Indeed, current 57 protocols (DNA extraction, library preparation, target-enrichment) are time consuming and 58 require handling expertise. They have been initially developed to work with large amounts of 59 input DNA (e.g., vertebrates or large/medium size insects; Faircloth et al. 2015; McCormack 60 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 62 on i) a large number of specimens/species to be representative of the overall diversity of the 63 group, without the possibility of using pipetting robots that increase DNA loss, ii) single 64 individuals because species complexes are frequent (Al Khatib et al. 2014; Kenyon et al. 65 2015; Mottern & Heraty 2014), iii) the whole insect without destruction for vouchering and 66 often prior to species identification, iv) rare species that have been collected once and may be 67 represented in collection by a few specimens or only one specimen and, sometimes, v) old 68 and dry museum specimens used for species description (types). 69 3 bioRxiv preprint doi: https://doi.org/10.1101/437590; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 70 In this study, we propose optimised protocols for DNA extraction and library preparation for 71 target enrichment purposes, as well as a custom pipeline to analyse the sequence data 72 obtained. We used these protocols and customised pipeline to capture and analyse Ultra 73 Conserved Elements (UCEs) in minute wasps, the chalcids (Insecta: Hymenoptera: 74 Chalcidoidea, Heraty et al. 2013; Noyes 2018), that are key components of terrestrial 75 ecosystems. Chalcids are key models for basic and applied research. With an estimated 76 diversity of more than 500,000 species these microhymenoptera have colonised almost all 77 extant terrestrial habitats. Many of them develop as parasitoids of arthropod eggs, larvae or 78 pupae. As such, they are both key regulators of the populations of many other arthropod 79 species in natural ecosystems and are increasingly used worldwide as biocontrol agents (e.g., 80 Consoli et al. 2010; Heraty 2009). A few of them, especially Nasonia (Pteromalidae) or 81 Trichogramma (Trichogrammatidae) species are also used as model systems to answer 82 challenging questions about sex determination, genetics of speciation, host-symbiont 83 interactions or behavioural ecology (e.g., Pinto et al. 1991; Stouthamer et al. 1990; Werren & 84 Loehlin 2009). Chalcidoidea has undergone a spectacular radiation resulting in a huge 85 diversity of morphologies and sizes (Gibson et al. 1999; Heraty et al. 2013), but are generally 86 small insects (< 2 mm long). Among them, Kikiki huna Huber (Mymaridae) at 158 µm long is 87 the smallest winged insect currently known and the wingless male of Dicopomorpha 88 echmepterygis Mockford at 139 µm is the smallest insect currently known (Huber & Noyes 89 2013). Notably, most species used for biological control, belonging mainly to five families 90 (Aphelinidae, Encyrtidae, Eulophidae, Mymaridae and Trichogrammatidae) are among the 91 tiniest wasps on earth (< 1mm). 92 93 Their small size, huge diversity and widespread morphological convergence make chalcid 94 wasps difficult to identify to species by non-expert taxonomists, which limits their use in 4 bioRxiv preprint doi: https://doi.org/10.1101/437590; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 95 biological control. Attempts have been made to resolve the phylogeny of the whole 96 superfamily (Heraty et al. 2013; Munro et al. 2011) or a few families (Burks et al. 2011; 97 Chen et al. 2004; Cruaud et al. 2012; Desjardins et al. 2007; Janšta et al. 2017; Owen et al. 98 2007) but none has succeeded. Indeed, the few markers that could be targeted with Sanger 99 sequencing were not informative enough to solve deeper relationships. A study based on 100 transcriptome data (3,239 single-copy genes) obtained from 37 species of chalcids and 11 101 outgroups also failed to solve relationships within the superfamily (Peters et al. 2018). As 102 only a representative sampling in both markers and taxa will allow one to draw accurate 103 conclusions on the history of this hyperdiverse group, target enrichment approaches appear 104 relevant. More specifically, targeting UCEs and their flanking regions that have been proven 105 useful to solve ancient and recent divergences (Faircloth et al. 2012; Smith et al. 2014) seems 106 pertinent. Indeed, a set of probes has been developed to target UCEs in Hymenoptera 107 (Faircloth et al. 2015). This set and an enriched one (Branstetter et al. 2017c) were 108 successfully used to solve the phylogeny of a few groups of ants, wasps and bees for which 109 the amount of DNA was not limiting (Blaimer et al.