Question: Phylogenetic tree construction with RAxML and low bootstrap values
1
gravatar for vasilislenis
4.6 years ago by
vasilislenis100
United Kingdom
vasilislenis100 wrote:

Hello everybody,

I have assembled the mitochondrial genome of 8 sheep individuals and now I am trying to construct the phylogenetic tree.

I have also downloaded from ncbi 2 representatives from each haplogroup. (So, I have 18 mitochondrial genomes in total).

With clustal omega I did the multiple alignments online and I used RAxML for the  generation of 500 ML trees.

I did bootstrapping (number of bootstrap replicates: 2000) but I'm not so confident with the bootstrap values.

Well, the topology seems ok, since all my samples belongs to haplogroup B and that is obvious from the tree, but the bootstrap values between them are low. 50, 48, even lower.

The bootstrap numbers at the major branches (the branches that categorizes the haplogroups) are very good (100, 98, etc). The problem seems to be in the small branches among my individuals.

I try to build a tree only with my individuals but again, I have some very good values (around 100) but also and some weak.

As it seems, I'm doing something wrong in my methodology but I cannot think what is.

Could you help me with the steps that I have to follow?

Thank you very much in advance,

Vasilis.

ADD COMMENTlink modified 4.6 years ago by 5heikki8.6k • written 4.6 years ago by vasilislenis100

Hey,

First thing I'd like to know is what are you aligning from these mitochondria? Is it their whole mitochondrial genomes, only some of their protein-coding genes or something else?

The alignment step is the one that will have the biggest impact on your ML generated tree, so I would suggest some manual QC of the alignment (and maybe try some other alignment programs as well) before jumping to downstream processing with RAxML.

ADD REPLYlink written 4.6 years ago by Cytosine450

Yes, I tried will the whole mitochondrial genomes.

I did the alignments with clustaw and muscle and the results were pretty the same. I used MEGA for this and manually I put one or two one base gaps to fix them. Actually seems pretty good.

What do you mean manually QC?

Do you believe that I have to restrict it in smaller regions?

Thank you very much

ADD REPLYlink written 4.6 years ago by vasilislenis100

You could try if only coding-regions will give you better resolution between samples, since those are more conserved and any variation in them is going to be more significant, than if you compare them along with intergenic regions.

ADD REPLYlink written 4.6 years ago by Cytosine450
1
gravatar for 5heikki
4.6 years ago by
5heikki8.6k
Finland
5heikki8.6k wrote:

I imagine that there's very little variance in the sheep mitochondrial genomes and that you might have to sequence 100's to 1000's of individuals to get nice bootstrap support values..

ADD COMMENTlink written 4.6 years ago by 5heikki8.6k

You are right, but unfortunately I have sequenced only 8. 

Unless if I will download more already assembled from ncbi.

ADD REPLYlink modified 4.6 years ago • written 4.6 years ago by vasilislenis100
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