Sequence comparisons (Bioinformatics?)

The ClustalW web site I linked to in the previous thread was chosen mainly because it has a relatively simple interface, the tree drawing was very simple to use and because it was pretty fast.Probably the most used portal to ClustalW is the one at Bioinformatics Tools for Multiple Sequence Alignment < EMBL-EBI which is hosted by the European Bioinformatics Institute. The EBI site has the full text of a paper which goes into some detail on the various alignment methods used by the ClustalW program.Anyone really keen might be advised to download a copy of ClustalX which should also allow you to try some bootstrapping, the download site has versions for a wide variety of OSs. The NJ-plot program is also downloadable and will allow you to fiddle around with the tree a bit more, displaying bootstrap values and branch lengths, or changing your outgroup if you have one.Some relevant highlights from the paper.On the distance matrix and pairwise alignments :- A further worthwhile exercise might be to get the corresponding nucleotide sequences for the proteins we have been looking at and run them through the same procedure.TTFN,WKThis message has been edited by Wounded King, 06-09-2005 06:13 AM

Here is a tree base on Cytochrome B protein sequence data from a number of species, including some insects, bacteria and a plant. I decided to use Arabidopsis as the outgroup since it was the only plant sequence I put in.As well as the sequences I already had for the marsupials I got a number of sequences by using the Homologene database on Entrez which pulls up homologues of a gene from a number of different species and will display multiple alignments of the protein products of those genes, and also will display them all in FASTA format making it very simple to import into Clustal.I then ran the data through a local version of ClustalX and used Treeview to produce the tree with the bootstrap values on.One downside to Clustal for bootstrapping, compared to a more dedicated program like those in the Phylip suite, is that it doesn't produce a record of the trees generated by the bootstrap. ClustalX will only add the bootstrap information to the tree while Phylip's seqboot program will actually generate a file containing the 1000 trees generated.The description of bootstrapping I gave in the previous thread was totally inaccurate. A more accurate description of the process is given by the author of the Phylip suite of programs:- In other words a dataset of-ACGTGCCTTGAGTGT
ACCTGGCTTGAAAGTMight become -AAACGTCCCTTTAAG
AAACCTGGCTTTAAAWhen the first column is sampled 3 times, the second once, the third once, the fourth once, the sixth twice, the seventh once, the eighth once, the ninth twice, the eleventh twice and the twelth once.311102112021000
_______________
ACGTGCCTTGAGTGT
ACCTGGCTTGAAAGT TTFN,WKedited by AdmiJar to downsize image.This message has been edited by AdminJar, 06-10-2005 12:17 PM

What molecular data? This is the absoloute rock bottom of scholarship, no reference, no suggestion what the molecule/s in question were. As far as evidence goes our scanr review is considerably more compelling than this utterly useless reference.But even were that not the case there are other published studies that support the marsupial mole being genetically closer to the other marsupials.Studies on the interphotoreceptor retinoid binding protein group Notoryctes with other marsupials, bear in mind that since the marsupial mole is blind this gene is virtually functionless in the marsupial mole. (Springer, et al., 1997).TTFN,WK

Please post the whole of the FASTA sequences, or at least provide sufficient information so that other people can get hold of the neccessary sequence data. In fact it might be a good idea if from now on people gave us the accession numbers of the sequences they run.One major problem here is that Cytochrome C is not 34 amino acids in length.I tried to run the same alignment using the following sequences As you can see the CytC sequences are 104aa in length. The sequence accessions are P68517 for the rattlesnake, P00022 for the turtle and P00014 for the Kangaroo.I'm not sure how you ended up with only 34 amino acids for each species, I'm not sure that you are using the FASTA format correctly. It looks as if you are losing all of the sequence which is in the first line of amino acids in my input data and only getting the last 34 amino acids.That aside the results are in line with yours. TTFN,WK

Here is the problem!Youre FASTA formatting is all wrong.The correct format is i.e. You need a line break after the name and only one '>' right at the start of the name for each sequence.At the moment you are losing big chunks of sequence data.TTFN,WK

It would be a very good idea to check out the source of the data in a case where there are severe discrepancies in the lengths of the proteins.A lot of the sequences in genbank are only partial coding sequences. For instance your 85aa sequence for the snapping turtle is only a partial coding sequence. There is a fuller, though still not complete, CDS based vesion here.This sort of partial data is bound to affect the quality of any analysis you perform. To check the source data for your amino acid sequence click on the hyperlink at the 'DBSOURCE' entry in the GenPept view.TTFN,WK

This is for Mark24 since the other thread in which we were doing sequence analysis has been closed.There is a windows version of ClustalX which should be compatible with XP, here. It comes bundled with the NJ-plot program as well, which you could use instead of Treeview.TTFN,WKThis message has been edited by Wounded King, 06-12-2005 09:07 AM

The easiest form of input is simply to make a plain text file with a set of FASTA data like those we have been using previously. You can get Genbank or GenPept to display the DNA/protein sequences in FASTA format and just c+P them into a txt file. You can even get a set of FASTA files throught the Homologene database.TTFN,WK

If people are really interested in bioinformatics then I would definitely recommend downloading the Phylip suite of programs. It is fairly fiddly and technical to use all of the programs to do exactly what you want but the analyses produced are considerably more powerful and sophisticated than the sort of things we have been doing with Clustal in terms of phylogenetics.TTFN,WK

Another nice new program has just been released. V2 of Jalview is out now and while it is not the best program for doing your actual alignments it allows you to visualise pre-existing alignments quite nicely and does some quite cool things with the trees, although it won't let me specify an outgroup for some reason. It also lets you run sequences through ClustalW and will display your data as a principal component analysis plot.Some quite nice features to play around with.TTFN,WK

*Bump*Just bringin this up to the surface since we have a number of new faces on the board at the moment, and also 'cos modulous was mentioning notbeing able to find it with search at the moment.TTFN,WK