Complete beginner trying to conduct phylogenetic analysis

[Tarka Dal]

I’m an undergraduate student that is trying to add some phylogenetic data to a lab report. Phylogenetic analysis wasn’t part of the taught content but my supervisor is happy for me to add it provided I make it relevant to the module content.

The organism that we examined was Trypanosoma brucei. My supervisor has recommended comparing it to T. cruzi, Leishmania tarentolae, Bodo saltans and Arabidopsis. I’m planning to include all three T. brucei subspecies in the analysis. I’ve managed to take some sequences from NCBI and have created a tree using PhyML, which seems to look OK.

I tried searching for 18S rRNA sequences on NCBI as they were the basis for a paper recommended by my supervisor (Stevens 1999). However, I was unable to find sequences for all of the species that were around the same length. From the reading I’ve done using sequences that have large differences in length is a bad idea. As such, I substituted 18S for an SSU rRNA sequence that was used in another paper I found on the topic. Stevens et al 1998 used M12676 when creating a tree containing the subspecies T.brucei.brucei. However, when I looked at NCBI it states that it's a T. brucei SSU and doesn't reference the subspecies it belongs to. I'm not sure if I'm missing something here? Additionally, when I do a nucleotide search for "trypanosoma brucei brucei" it produces mRNA and DNA results but no rRNA results, which seems odd.

I have the following questions:

1) Am I doing something stupid when searching for sequences? It seems odd that I can't find any rRNA sequences for T. b. brucei and I'm not sure why M12676 is defined as an T. brucei SSU when some papers have narrowed it down to T. b. brucei.

2) Are there any other databases I should search in apart from NCBI?

3) The paper the project was based on contained a diagram showing which surface proteins are expressed in different eukaryotes. The proteins the genes of interest (Tb927.8.6720, Tb927.7.4230 and Tb927.8.6730) code for are specific to trypanosomes and closely related parasites. As such, creating a multi-species tree for the genes isn’t going to be practical due to a lack of orthologs. Does anyone have any ideas on how I could link this in to the work I’ve already done on the species tree? I was considering replicating the work done in the paper and creating a phylogenetic tree for the species referenced so that you can visualise how closely related the relevant species are. Anyone have any better ideas?

Thanks in advance for any responses. Nothing is too basic for me at the moment. Sorry if this is a bit long winded.

[avierstr]

Hi Tarka Dal,

NCBI is not consistent in the naming of the genes. Sometimes they use SSU, sometimes 18S for the same gene. Sometimes they use rRNA or also DNA. In fact the sequences are the same. So I would advice to look for "Trypanosoma brucei AND 18S" and an other search for "Trypanosoma brucei AND SSU".

An other possibility is to use BLAST. Take one of your sequences and blast them. BLAST will return the most closely related sequences it has in the database that you can use for your tree.

https://blast.ncbi.nlm.nih.gov/Blast.cgi

Your question 3 seems a nice idea. I suppose you will use 18S to make that tree?

Greets,
Andy

[Tarka Dal]

Thanks for the info Andy. Good to know that NCBI isn't consistent, I've been tearing my hair out for a while wondering why some of my results seemed a bit odd.

Going with 18S seems to be the best idea as it's been used in some of the papers that my supervisor has recommended.