Illuminating the firefly genome

Hi Misha, sorry for the late reply. I received the email on this comment but was unable to track down exactly where it was... For other fireflies, if people really want to they could use the P.pyralis genome to scaffold, but beware as indeed there is no guarantee that genomic structural organization is well conserved in fireflies. Data on the firefly repeats from the P.pyralis genome could be beneficial for assembly by enabling masking. The genome size is a definite issue. Sarah Sander has data to demonstrate there is large (very large) variation in firefly genome sizes (luckily P.pyralis is smallest!), I'd touch base with her if you are curious about a particular species. I think the biggest benefit of a P.pyralis genome will be to serve as reference to help evaluate how other firefly correct assemblies are at a local scale (as relatively minor changes in assembly procedure can drastically affect final assembly results)

The background signal for luminescence (autoluminescence) is much lower than the background signal for fluorescence (autofluorescence), so luminescence does end up being more sensitive in many applications.

Hi Misha, for an Illumina de-novo genomic strategy strategy one could do PCR-free library prep & HiSeq 2500/4000 250x250 PE sequencing + DiscoVar DeNovo assembly, for around 1-5ug of DNA, which from our experience with an optimized prep is at the edge of possible from 1 large specimen of P.pyralis, but more reasonably is around 10-20 fireflies to be safe. I think Photuris are generally a similar size to Photinus pyralis so the projection is likely similar. Keep in mind though that Illumina won't perform as well as long-read PacBio for de-novo genomic assembly, and although it is "cheaper" / requires less DNA, when you get down to the most recent cost projections PacBio is actually pretty competitive, and gives genomes of beautiful quality. You also need more sequencing/ input DNA (roughly proportionally) for the genome size, which can vary massively even within a single taxonomic family. In our case since we're going for PacBio, we needed high-molecular weight DNA, which is tougher to extract, so we're currently looking at DNA extraction from the order of 100 fireflies, though we hope ongoing optimizations can reduce that number down as when sequencing from a wild population heterozygosity is a big issue for the assemblers. We plan to post more information on "why PacBio" and the technical details of the early stages of the genome project later this week, as we know its something that people are interested in, but we sort of ran out of time/space with the initial description for the project : ) I wish we could grow fireflies! As far as I know there are no cell-lines for any North American fireflies, and there aren't lab-rearable Luciola fireflies available to us here in the US.

Hi Misha- Thanks for your many questions! Since you're interested in many of the more technical details of our methods (which we appreciate!), perhaps it would be best for us to answer these technical questions via email. Please feel free to contact any of the team directly. Many thanks for your interest in this project!

Hi Misha, Calibrated flow cytometry with propidium iodide stained nuclei is a standard method. Here is a good citation: Hare, E.E., and Johnston, J.S. (2012). Genome Size Determination Using Flow Cytometry of Propidium Iodide-Stained Nuclei. In Molecular Methods for Evolutionary Genetics, V. Orgogozo, and M.V. Rockman, eds. (Totowa, NJ: Humana Press), pp. 3–12. It is relatively cheap, if you have access to FACS facilities. Alternatively, its possible to estimate genome size from Kmer analyses of high-throughput sequencing data, but its probably not "acceptable" by itself as a measure of genome size. Many of the genome assemblers will do this type of Kmer analysis and report an estimated genome size while doing assembly.

Hi Misha, Our genome size estimate was obtained with flow cytometry of propidium iodide stained nuclei from neural tissue in collaboration with Spencer Johnston at Texas A&M (see Tim's link below).

Hi Misha, I'd say Photinus pyralis is scientifically one of the most well-studied firefly species as it is one of the most abundant & widely distributed species. In terms of scientific study, the European glowworm Lampyris noctiluca & the Japanese Genji firefly Luciola cruciata is also pretty good, but among those candidates, P.pyralis is our local favorite!