For anyone wanting to learn or teach basic phylogenetics for DNA barcoding and identification, here are three introductory tutorials for identifying fungi, tardigrades, and flies, all using the free MEGA package for molecular evolutionary genetics analysis. All three tutorials cover essentially the same processes but are slightly different, so you could compare, contrast, find the one that works best for you, or combine the most useful parts of each! 🍄 The first resource, by Casanova et al. (2021) is an article describing three 2.5 hr lab practicals involving a mystery fungal specimen. It covers DNA extraction using a spin column kit, amplification of the internal transcribed spacer (ITS) barcode region for fungi, and sequencing. The mystery fungus sequence is then identified using BLAST searching in GenBank and MEGA is used to produce a maximum parsimony phylogenic tree. Supporting files can be found in the supplementary data. If you want to try this yourself without a spin column kit, it may be useful to know our Dipstick DNA Extraction Kit works very well for fungi and only takes a minute or less for extraction. Suárez Casanova, V. M., & Shumskaya, M. (2021). Exploring DNA in biochemistry lab courses: DNA barcoding and phylogenetic analysis. Biochemistry and Molecular Biology Education, 49(5), 789-799. iubmb.onlinelibrary.wiley.co… ⭐The second guide is a detailed protocol for tardigrade barcoding, by D’Elia et al. 2023. It describes how to find, sample, extract DNA using a spin column kit, and amplify the 18s rRNA barcode region using tardigrade-specific primers. It then uses the DNA Subway online portal (by the DNA Learning Center, Cold Spring Harbors) to produce consensus sequences; uses BLAST searching in GenBank for an initial identification and download reference sequences, and finally MEGA to construct a multiple sequence alignment and phylogenetic tree to analyse the sequences. For those without access to spin column kits and tardigrade-specific primers, you can also extract tardigrade DNA using the HotSHOT DNA extraction method (reducing the volumes to 20 µL), and use universal COI primers for barcoding. Additionally, if you want to work through the analysis part without doing the DNA extraction and PCR, you could use the sequence here as the mystery sample: ncbi.nlm.nih.gov/nuccore/MH6… D'Elia et al. 2023. A Complete Guide to Tardigrade Isolation and Phylogenetic Characterization for Undergraduate Students. protocols.io dx.doi.org/10.17504/protocol… 🪰 The third guide, by Lorusso et al. (2022), is a remote exercise working only with phylogenetics, which aims to guide a learner in the forensic sciences in constructing phylogenetic trees to identify unknown specimens. In this case the mystery specimen is a fly associated with a decomposing body (because different flies are associated with different decomposition stages), but exactly the same process can be used for biodiversity surveys. This exercise has a walkthrough of searching a COI barcode sequence in the BOLD database for an initial identification, followed by constructing a multiple sequence alignment and a phylogeny in MEGA, after which the initial identification can be reassessed. Most of the exercise information is in the downloadable teaching materials, consisting of a Powerpoint presentation, a student handout with instructions, and reference sequences. Lorusso et al. (2022). Applying phylogenetic tree building in MEGA X to forensic applications for identifying unknown specimens. (Version 2.0). QUBES Educational Resources. doi:10.25334/XY5F-XQ54 qubeshub.org/publications/25… If you find any of these learning resources useful, or if you can share other similar examples, please do let us know!

For anyone looking for a simple, rapid, non-toxic genomic DNA extraction method, here’s a classic method that has over 3760 citations and is still widely used today. It was originally designed for plants but has occasionally been used for animal or fungal DNA, and has many variations on the same theme, including one super-simple formulation that may be useful for amateur or educational use. The method, by Edwards et al. (1991), involves manual grinding followed by adding an extraction buffer containing the detergent SDS and NaCl (for cell lysis) in a Tris-EDTA buffer (to protect the DNA). It could take as little as 15 minutes per extraction, as below: ⭐Grind tissue with a plastic pestle without buffer ⭐Add 400 µl of extraction buffer (200 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), and vortex or mix. These samples can be left at room temperature while other extractions take place ⭐Centrifuge for 1 min at top speed to pelletise any solids ⭐Remove 300 µl of the supernatant into a new tube ⭐Add 300 µl of isopropanol to the new tube and mix to precipitate DNA, ⭐Incubate for 2 mins at room temperature ⭐Centrifuge for 2 mins at top speed to pelletise the DNA ⭐*Remove the isopropanol carefully with a pipette, allowing the residual isopropanol to fully evaporate (note: in the original protocol this was evaporated using vacuum drying) ⭐Resuspend the DNA in 100 µl of 1x TE buffer Here’s the article: Edwards et al. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic acids research, 19(6), 1349. ncbi.nlm.nih.gov/pmc/article… However, it’s worth bearing in mind that this is a relatively crude DNA extraction method, and that it may not work well with many plants (or other organisms) if they contain high levels of polysaccharides, secondary metabolites, or other PCR inhibitors. Although this method was designed for plants, it’s also been used for genomic DNA extractions from fish spleen and kidney, again incorporating a final 70% ethanol wash: Subramaniam et al. (2014). Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia. Journal of Fish Diseases, 37(7), 609-618. academia.edu/download/354087… And it has been used as-is or modified for use with fungi. For example, Rodrigues et al. (2018) used essentially the same protocol and reagents for fungal mycelium and conidia, incorporating an additional sodium acetate protein precipitation step and a final 70% ethanol wash: Rodrigues et al. (2018). Toxic reagents and expensive equipment: are they really necessary for the extraction of good quality fungal DNA?. Letters in applied microbiology, 66(1), 32-37. bibliotecadigital.ipb.pt/bit… To make this method high-throughput, you could use the modifications of King et al. (2014), who described a high-throughput DNA extraction method for soybean leaf and seed chips, using bead-beating, a 30-minute incubation at 60 °C, and a final 70% ethanol wash: King et al. (2014). Non-toxic and efficient DNA extractions for soybean leaf and seed chips for high-throughput and large-scale genotyping. Biotechnology letters, 36, 1875-1879. researchgate.net/profile/Zac… And finally, for an even more bare-bones approach, Mangam et al. (2010) described a variation of this method for use with insects, removing EDTA and Tris-HCl from the extraction buffer, increasing the NaCl and SDS concentrations, and again using a final 70% ethanol wash: Margam et al. (2010). A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding. Molecular biology reports, 37, 3631-3635. researchgate.net/publication… Margam et al. (2010)'s method could also be very useful as a very accessible approach for community science, educational, or resource-limited work, since it uses only SDS, NaCl, isopropanol, and ethanol. Overall, it’s a very simple, safe, low-cost genomic DNA extraction method; easy to adapt to many sample types; and it appears to be sufficiently successful that it is still in use today. It won’t work with everything, and will probably work best with sample types with minimal PCR inhibitors. But even so it could be a good method to try if you’ve got a small budget and lots of samples. And if you use a version of Edwards’ et al. (1991)’s method already, please let us know — we would love to hear what you use it for! *** ncbi.nlm.nih.gov/pmc/article…