Kindly tell me why I am getting this message in Mega 6 software while constructing a phylogenetic tree.
I have created a meg. file having my sequence along with 4 related sequences from genbank. but when I try to open this file in MEGA 6, it says "Aligned sequences must be equal lengths (in line 28). The software is easily opening its "example file named hsp20.meg"
I have attached an image of the error message, kindly help me. I shall be highly obliged.
The Nucleotide sequences don't have to be exactly the same length, but they need to be aligned and the sequences that are shorter need to be "padded" with gap characters "---" so that the strings of characters following each sequence name are all exactly the same length. Many multiple sequence alignment programs (MAFFT, MUSCLE, CLUSTAL, T-COFFEE, etc) will do the alignment and also make sure that the resulting alignment has gap characters added to the ends where needed.
It will happen if u have not aligned the sequences. First align them using options in mega and then cut the ends and make them of equal size. Later on perform the phylogenetic analysis
Look at the message of MEGA - you can see that your sequences have different length - L. donovani has 9 nucleotides in the last line whereas the previous sequence has only 7.
:-)
Hi Sidana!
I'm facing same problem! You have solution or still waiting
Yahya
Muhammad, as told above, the problem is that the sequences are not aligned. You can see that they both end with AACCCAT but one of them has two more bases on that last line than the other one does. Somewhere, one of the sequences suffered a 2 base insertion relative to the other one.
I was having the same issue. as B.A.Padder suggested you can use MEGA to align the sequences. I had originally converted a FASTA file format (which I had exported from Geneious) into a MEGA file and this would not align due to the same sequence length issue. BUT you can use the alignment function on the original FASTA file. This allows you to create another file whereupon the alignment has been done and every sequence has the same length (as the gaps have been filled with -----). Then perform the phylogenetic analysis.
Hope this helps
I was facing the same problem, as i was using BioEdit for Clustal W multiple alignment. I think there is some problem in doing multiple alignment with this program. However when i used Mega 6 instead, my problem was solved.
I would like to suggest not to use BioEdit for the alignment of your sequences, you can just convert your fasta fomat text file in BioEdit to aln file format (without aligning it), once you have that aln format file, you can simply perform alignment like Clustal W or MUSCLE alignment Using Mega6 program. once you have the aligned Mega6 file format, then you can easily make the phylogenetic tree using the same program.
Dear Khushwant Singh
There isn't any "Retrieved from sequence" option in align menu!
A thin bold line passes through the file (as you see it when you open in MEGA), and the file and sequence characters need to be on the left hand side of the file. Generally, the file headers are longer, need to be trimmed with in the LHS space!
I encountered this problem when trying to align a downloaded blast search result (fasta format in .txt converted to .mega). For me simply manually changing the downloaded file extension from .txt to .fasta solved the problem.
Trick is to save first in aligned format with clustalW or MUSCLE and then close the session and open recently used file when the whole file will be displayed...then do your phylo trees. if you have further doubts let me know.
Cheers,
Bruno
I just encountered similar problem now on mega with error message: Symbol expected in line 1.
Fortunately, I solved the problem by changing the file from .meg back to .fas and it worked.
Cheers.
i spend one day almost, handling the same issue. my suggestion to you will be; cut the sequence and paste it somewhere else in same notepad file and convert it from .txt into .fas file . As spacing issue may be resolve. I have also solve my problem the same way.
better to use MEGA-X
Thanks
Hi, sometimes this will happen because you have symbols that are not allowed in your fasta headers - remove any =, ", etc in your alignment/fasta file headers and you should be good.
Hii..
I got such error message and sorted it by using the session file to use it for either model testing or phylogenetic tree contruction instead of .mega file
.
Redownload all sequences in batch and align them in MEGA. Avoid editing them
as much as possible.
Just had the same error. I fixed by saving the alignment as fasta instead of MEGA format,
MEGAX gives the same error. My experience is little different. Following these initial steps may be helpful:
Align> Edit Build New Alignment> In alignment editor select "Create New Alignment" : Alignment window will be opened then, copy your sequences (make sure no spacing problem is exist!) and paste into the new window. Do not forget to delete additional row opened during the Alignment Window. The align and export to the folder and follow other steps in given above...
the trick that always worked for me was to trim the sequences after muscle alignment by selecting through the left mouse key from the top of the panel and then right-click you will find a delete pop up appears in mega from both ends until all sequences are equal and then save it as a .meg file.
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