Reddit reviews Phylogenetic Trees Made Easy: A How-To Manual

OK, well since you already know what species you're interested in, your next step would probably be to choose a gene to use. So, take your organisms of interest and see what's available on GenBank for them. It looks like the choroplast rbcL gene might be a good choice - it seems to be a barcode gene and there are multiple specimens of Avicennia germinans and Rhizophora mangle available on genbank. Unfortunately, it doesn't look like anyone's sequenced Maytenus phyllanthoides yet, but there are some Maytenus segovarium rbcL sequences on GenBank, so that might be a good substitute. Another approach would be to search for papers on the phylogeny of eudicots and see what genes authors in your area use - it tends to vary among different organisms, but usually there will be one or two widely sequenced genes. You can also combine two or more genes together for a more robust phylogeny. At this stage, I would probably search for the gene name "rbcL" (sometimes people use different names for the same gene, but GenBank usually knows all the synonyms, to be safe though, you can try searching for alternate names also) and the group I'm interested in e.g. "eudicotyledons" (you might call this group something different, but GenBank's naming system is very conservative, and you've got to use the name GenBank recognises). Now, that seems to have turned up around 47,000 so this approach probably won't work very well here, but I'd normally just download everything and then trim off the ones I don't want later. Some of the results will be different specimens of the same species, some might not be the rbcL gene, or at least not the part of it I want. There might also be a mix of complete genes and partial genes, but this is OK. It looks like there's a lot of partial rbcL genes that are exactly 583 bp long - they're almost certainly the same section of the same gene. Some are a bit shorter (e.g. 549 bp)- that could be due to actual deletions in the sequence or the use of different primers by the people who sequenced it. It could also mean that it's a different section of the gene, but that's fairly unlikely and should be clear when it doesn't align to the sequences of the other species - or to a different section of any complete genes.

So, after you've chosen your species and your gene/s you need to start inferring phylogenies. For help with that, I'd suggest seeing if your library has a copy of Phylogenetic Trees Made Easy.

I have experience with MEGA to generate trees. I agree that it's a clunky software, but once you understand how to use it, it's not so bad.

I recommend the following book if you want to become a MEGA power-user: https://www.amazon.com/Phylogenetic-Trees-Made-Easy-Manual/dp/0878936068


Before you start... you say you are looking at the protein sequences. Are you starting with the amino acid sequence or the mRNA sequence? If mRNA, make sure all sequences are in the same orientation (ATG/start on the 5' end of the sequence). I find that if you have just one or two sequences that have been copied in the reverse direction, the alignment may not work. You can also save multiple alignments -- try some with seemingly more conserved sequences (less gaps) and try the entire set.

Once you have all your sequences in one FASTA file or multiple FASTA files, import them into MEGA. From the 'Align' menu, choose to edit/build an alignment. Add your sequences. Select all sequences for alignment and then align selected (you can get into some advanced settings here, change the alignment algorithm, etc.) Save the alignment as a .mas file and export as a .meg file.

Now, open your .meg file in MEGA's main window. From the phylogeny menu, select to build a neighbor joining tree. Based on the alignment, the program will auto-build a tree for you. Again, lots of functionality exists in the program, but it's not an intuitive UI.

Happy to help if you have other questions!

Barry Halls Phylogenentic Trees Made Easy Link