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Determining genome size: one step closer to sequencing whole genomes of important biocontrol species
ESR Update - One of the first steps for any sequencing project for a non-model organism is to determine its genome size – about how many base pairs does your organism have? This directly impacts costs of sequencing as well as an accurate estimation of coverage. There are three species within RP1 and only one them has a known genome size: Trichogramma brassicae, with an estimated genome size of 246 Mb. So, the next step in my project is to perform flow cytometry on the remaining species.
Kim Ferguson MSc.
I’m analysing Amblyseius swirskii and Nesidiocoris tenuis, as well as Trichogramma brassicae and Trichogramma evanescens. This is alongside a Drosophila melanogaster reference and various controls. Timing is everything for this kind of work: machines are booked in advance, stains have a window of efficacy, and material needs to be fresh. All of the prep work was performed the day before (as pictured), followed by staining the next day before being analysed.
The work itself is on a small scale: from one organism, the head is removed, gently crushed, and extracted nuclei are stained. Then it’s over to the flow cytometer where individual cells are excited by lasers and their fluorescence detected. The eventual output is in the form of graphs where differences between the positive controls, references, and our specimens are compared for ploidy, number of nuclei, and eventually genome size.
The results from this experiment are key: once I know genome size of all my species, I’ll be better equipped to finalize a sequencing strategy, as well as be able to test rearing methods on the different species before beginning the precise work of rearing and creating iso-female lines. In the end, I didn’t get all the results I needed in the first go, either due to chance, skill, or a necessary tweak in my protocol, but I’m getting there, and should have reliable and beneficial results soon.
It sounds like I’ll be doing this solo, but that’s far from the truth. Specimens were collected from Koppert BV as well as Nina Fatouros in Entomology here at Wageningen. One of my supervisors here in the lab of Genetics, Eveline Verhulst, assisted with prep and staining, while Marcel Tempelaars in the lab of Food Microbiology performed the flow cytometry. Additional thanks to fellow ESRs Kelley Leung and Sophie le Hesran for their help in collecting tools and specimens, Kirsten Oude Lenferink at Koppert BV for assistance with the swirski mite, and Jetske de Boer at the lab of Entomology for her assistance.