Dear friends... Im doing project on evolutionary relationship and pathway reconstruction of anthocyanin pathway on solanaceae family plants... for that i collected 13 group of gene sequences(850 seqs.) ... i felt very tough in multiple sequence allignment step because of more number of sequences... how to prepare the Multiple Seq. Alignment for phyogen. tree construction?? i got error when using MUSCLE in mega software... in clustalw i got result but there are many coloumn of sequence alligned... how to manually allign them?? hoe i select columns for phylogenetic tree construction?? thank you..
Well, when you are working with so many sequences my advice is:
1. Do an alignment in MUSCLE but implemented in a webpage not in MEGA. I don't know how it works in MEGA.
2. After that using the amino acid alignment load the data in JavalVIew and do a first visualization.
3. Try to improve your alignment taking in to account physical and chemical properties of the amino acid (check Jalview manual).
4. Use MEGA to check nucleotides alignment and remove indels or regions that you are not sure that they are homology nucleotides. I mean regions that are not well aligned.
5. Do phylogenetic analysis.
I hope it helps you as a preeliminary advice.
P.S: Scripts or basic knowlegde of Python or Perl can help you to manipulate your data.
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