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Aligning 454 data to a reference sequence (Read 11563 times)
Mar 6th, 2012 at 11:44pm

TSchneider   Offline
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I have been trying to align my 454 data to a reference sanger sequence in UGENE, but I just can't seem to figure it out. All I want to do is look at intra-individual diversity levels of virions within hosts. Basically, I just need to know what sequences are present, how many there are, how different they are from my original sanger sequence, and the percentage of each sequence within the host.

I think I might have to do an assembly, however every time I try (I've tried both Bowtie and UGENE aligner) it doesn't work. Am I picking the reference sequence wrong? How do I get this to work?? It sounds exactly like what I need.

What I do, is I have my 454 data file in the program. I select it then go to DNA assembly. Then where it asks for the reference sequence, I direct the program to my sanger sequence file. Then I click run without changing any parameters, but it hasn't worked.


Any help is appreciated! Thanks  Undecided
 
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Reply #1 - Mar 7th, 2012 at 3:03pm

Yuriy Vaskin   Offline
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Thank you for the report and interest in our project.

You do it right to align your data. What message does UGENE show you when you click the align button?

Also, it might be a problem with data formats. What are the formats of files with your reference sequence and short reads?
 
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Reply #2 - Mar 7th, 2012 at 4:23pm

kenkl   Offline
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Hi, TSchneider I think that should be due to the incompatibility of the data format of the 2 sequences that you wish to align. Raw data from 454 is SFF files whereas Sanger reference is AB1 file. I suspect that should be the reason.

 
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Reply #3 - Mar 8th, 2012 at 12:45am

TSchneider   Offline
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I've put both files into fasta format before uploading them to UGENE...
 
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Reply #4 - Mar 8th, 2012 at 1:18am

TSchneider   Offline
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I believe that the option to "Align sequences with a reference file" when first opening my files in UGENE sounds exactly what I need to do. However, whenever I upload my file and click that option-- it doesn't work. Nothing is even uploaded! There is no error message of any kind shown either.
 
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Reply #5 - Mar 12th, 2012 at 3:01pm

Yuriy Vaskin   Offline
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Dear TSchneider, we apologise for the response delay.

The problem is that the "Align reads to reference sequence" option you used from the "Sequence reading option" dialog doesn't work yet. I've created an issue (https://ugene.unipro.ru/tracker/browse/UGENE-882) in our bug-tracker which will be fixed in the next UGENE release.

You can perform the alignment by launching the "Align Short Reads" dialog from the "Tools" menu ("Tools->DNA Assembly->Align short reads..."), specifing your reference sanger sequence and adding 454 reads (all in FASTA format). Moreover, we've sent you a detailed description of the process on your email.

Please, try to do the alignment in that way and if you still face any problems, feel free to contact us.

Also, if there is still an error, it will be easer to fix it if you send us the test files.
 
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