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Error when using map Sanger data analysis (Read 299 times)
Sep 28th, 2020 at 10:35pm

Mateo   Offline
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Hello everyone,

I am quite new to the program, but tutorials on youtube and topics on this forum were quite helpful to get this far.
So, I have ab1 files with forward and reverse strand (different primers). I managed to get the reverse complement, find the consensus, export it and in final step (just like in this video: "Unipro UGENE podcast #52: The Sanger Reads Editor in UGENE 1.27") I get the following error: ugene Error: Subtask {Compose alignment} is failed: No read satisfy minimum similarity criteria.

Really don't know what am I doing wrong. In the input data I put my consensus file in "reference" tab, and in "reads" I add those two aforementioned ab1 files with different primers. Everything else is by default (Trimming quality threshold 30; Mapping min similarity 80).

Thanks in advance guys Smiley
Mateo
 
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Reply #1 - Sep 29th, 2020 at 12:18pm

Dmitrii Sukhomlinov   Offline
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Hello Mateo,
The problem is that reads could be filtred if the similarity percentage is lower than the value you've set up (the "Mapping min similarity" parameter) - more details here https://ugene.net/wiki/pages/viewpage.action?pageId=49447480&from=ugene . You need to decrease this parameter before the run.
To make sure, you can check the report - click on the notification on the right bottom corner after sanger mapping if finished.
 

Best regards,
Dmitrii Sukhomlinov,
UGENE team.
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Reply #2 - Sep 29th, 2020 at 4:38pm

Mateo   Offline
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Hello Dmitrii,

Thank you so much! After I tried your solution it worked. However, I adjusted "Mapping min similarity" to 0%. Can there be a problem with putting that low of a percentage? In other words, is it more advisable to decrease that parameter by smaller margins (say 10% at a time) and figure out what is the first percentage that would work?
 
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Reply #3 - Sep 29th, 2020 at 6:09pm

Mateo   Offline
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Btw, when I go to next sequences and do the same work I get the same error even with adjusted percentage... I tried with every 10th% (0-99%) and it always returns same issue. How can I know what is specific Mapping min similarity for each of my data files?
 
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Reply #4 - Sep 30th, 2020 at 1:14pm

Dmitrii Sukhomlinov   Offline
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Hello again,
As I've already said, after every "Trim and map Sanger reads" run you've got the notification in the right-bottom corner - blue or red, depends on whether the execution went good or not. You may click on it and the execution report will be opened.  In this report, you may find the information about similarity. Try to check it and you will see, what value you need.
 

Best regards,
Dmitrii Sukhomlinov,
UGENE team.
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Reply #5 - Sep 30th, 2020 at 4:53pm

Mateo   Offline
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Thanks for reply once again. But the problem is, I don't see any value. When I click on red notification all I get is just the issue:


"Task report [Map Sanger reads to reference]




Status
Failed
Error:
Subtask {Compose alignment} is failed: No read satisfy minimum similarity criteria.
Time
0h 00m 04.228s"

I will try to put attachment with screenshot here...
 
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Reply #6 - Oct 2nd, 2020 at 10:14am

Dmitrii Sukhomlinov   Offline
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Hello again, sorry for waiting,
There are signs in the log "read_name was skipped. No BLAST results". That means that the BLAST tool, which looks for the approximate place in the sequence, where the read should be put on, couldn't find any region for this read. In general, reads you've tried to align aren't fit to the reference at all.
 

Best regards,
Dmitrii Sukhomlinov,
UGENE team.
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Reply #7 - Oct 5th, 2020 at 5:29pm

Mateo   Offline
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No problem, I am glad you answered after all. And, btw, thx for making all this effort. However, I have to ask final question regarding this. What exactly does your reply mean? I've put my sequences against reference (consensus). Is it my fault, meaning I selected wrong files? Or, is something wrong with my sequences (reads)?
 
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Reply #8 - Oct 6th, 2020 at 11:02am

Dmitrii Sukhomlinov   Offline
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The idea is that your read and reference are too different to find any way to align them with each other. If you send me your data, I'll take a look at them and, maybe, it'll help me to tell you more information.
 

Best regards,
Dmitrii Sukhomlinov,
UGENE team.
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Reply #9 - Oct 15th, 2020 at 5:35pm

Mateo   Offline
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Sorry for taking so long to reply, I was busy with some other work. Anyway, I would upload files for you here but they are bigger than it is allowed...
Anything we can do to solve this?

Mateo
 
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Reply #10 - Oct 16th, 2020 at 10:47am

Dmitrii Sukhomlinov   Offline
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You can download them, for example, on google drive and send me a link to the folder.
 

Best regards,
Dmitrii Sukhomlinov,
UGENE team.
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Reply #11 - Oct 16th, 2020 at 5:15pm

Mateo   Offline
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Reply #12 - Oct 19th, 2020 at 2:00pm

Dmitrii Sukhomlinov   Offline
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Yes, I can. I downloaded files and now I can see. The reference sequence and reads aren't looked similar so it's not surprising that mapping doesn't finish well. BLAST just couldn't find a place on the reference where to put the input reads. I think, you need another reference sequence.
 

Best regards,
Dmitrii Sukhomlinov,
UGENE team.
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Reply #13 - Oct 19th, 2020 at 4:19pm

Mateo   Offline
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Hmmm, ok... I'll play around with some other sequences and see if those are any better. Thank you very much for all your work and help.

Cheers!
Mateo
 
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