Pushing the limits of whole genome amplification: successful sequencing of RADseq library from a single microhymenopteran (Chalcidoidea,Trichogramma)

Abstract

A major obstacle to high-throughput genotyping of microhymenoptera is their small size. As species are difficult to discriminate, and because complexes may exist, the sequencing of a pool of specimens is hazardous. Thus, one should be able to sequence pangenomic markers (e.g., RADtags) from a single specimen. To date, whole genome amplification (WGA) prior to library construction is still a necessity as at most 10 ng of DNA can be obtained from single specimens (sometimes less). However, this amount of DNA is not compatible with manufacturer’s requirements for commercial kits. Here we test the accuracy of the GenomiPhi kit V2 on Trichogramma wasps by comparing RAD libraries obtained from the WGA of single specimens (F0 and F1 generation, about1 ng input DNA for the WGA (0.17–2.9 ng)) and a biological amplification of genomic material (the pool of the progeny of the F1 generation). Globally, we found that 99% of the examined loci (up to 48,189 for one of the crosses, 109 bp each) were compatible with the mode of reproduction of the studied model (haplodiploidy) and Mendelian inheritance of alleles. The remaining 1% (0.01% of the analysed nucleotides) could represent WGA bias or other experimental/analytical bias. This study shows that the multiple displacement amplification method on which the GenomiPhi kit relies, could also be of great help for the high-throughput genotyping of microhymenoptera used for biological control, or other organisms from which only a very small amount of DNA can be extracted, such as human disease vectors (e.g., sandflies, fleas, ticks etc.).

Disjunction between canola distribution and the genetic structure of its recently described pest, the canola flower midge (Contarinia brassicola)

“…This is concordant with the findings of de Medeiros & Farrell (2018), who found that samples with less input DNA were more prone to reduced genome coverage after sequencing. Our results differ from those of Blairet al (2015) and Cruaud et al (2018), who both found negligible differences in genome coverage and sequencing depth when comparing WGA and non-WGA samples. However, we note that Blair et al (2015) used much higher quantities of input DNA for WGA than our study system permitted, and Cruaud et al (2018) pooled individuals so they were unable to make the same individual comparisons presented here, and in de Medeiros & Farrell (2018).…”

“…Reported differences in sequencing depth between treatments did not appear to impact de novo locus construction and SNP calling in the WGA test dataset, which was consistent with other studies (Blairet al 2015;Cruaud et al 2018;de Medeiros & Farrell 2018). Pairwise F ST comparisons, observed heterozygosity, and PCA indicated little difference in genotyping between treatments when they were filtered together (Appendix 1: Table A3, A4; Fig.…”

“…The development of whole genome amplification (WGA) techniques, which amplify genomic DNA prior to NGS library preparation and sequencing, present a possible solution to this problem. However, the application of WGA in NGS datasets is relatively new and there have been few studies to date that have assessed whether WGA is likely to introduce amplification biases that may impact genome coverage and genotyping, particularly in organisms that lack a reference genome (Lovmar et al 2006;El Sharawy et al 2012;Ellegaard et al 2013;Cruaud et al 2018). In the first study to comprehensively investigate WGA for insect population genetics using non-pooled samples, de Medeiros & Farrell (2018) found WGA resulted in sufficient libraries for analysis, albeit with fewer loci.…”

Optimized DNA extraction and library preparation for minute arthropods: Application to target enrichment in chalcid wasps used for biocontrol

“…Interestingly, as it has been shown previously on RADseq data (Cruaud et al, ), WGA does not seem to bias the results even when input DNA is below the recommendation on the manufacturer kit (here 0.4 and 4 ng when the GenomiPhi Kit V2 requires 10 ng). It is noteworthy that, after completion of this paper and using the same protocol, we were able to capture ca 300 UCEs from native DNA with a concentration that falls below the detection limit of the Qubit, without WGA.…”

Hybrid Genome Assembly and Evidence-Based Annotation of the Egg Parasitoid and Biological Control AgentTrichogramma brassicae

“…Field trials have been conducted on several aspects, such as host location and dispersal behaviour (Suverkropp et al 2009(Suverkropp et al , 2010, overwintering ability (Babendreier et al 2003), while other biological control related studies considered issues related to low temperature storage (Lessard and Boivin 2013), reaction to insecticides (Liu and Zhang 2012;Delpuech and Delahaye 2013;Ghorbani et al 2016;Jamshidnia et al 2018;Thubru et al 2018), or risk assessment (Kuske et al 2004). Next to its application as a biological control agent, this tiny parasitoid has been used in other research, both in genetic studies (Wajnberg 1993;Laurent et al 1998;Cruaud et al 2018) and ecological studies (Huigens et al 2009;Fatouros and Huigens 2012;Cusumano et al 2015). In addition, several initiatives investigate the infection of T. brassicae with Wolbachia bacteria (Poorjavad et al 2012;Ivezić et al 2018) and the consequences of such an infection (Farrokhi et al 2010;Poorjavad et al 2018;Rahimi-Kaldeh et al 2018).…”