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Message started by Bernie on Jul 16th, 2019 at 12:20am

Title: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 16th, 2019 at 12:20am
Hello,

I am a German mycologist and new to this forum.

My problem: I want to create the consensus sequence from a forward and a reverse chromatogram.
To do this I start with Tools -> Sanger data analysis -> Reads de novo assembly (with CAP3)...
But when I add the two chromatograms with "Add... > Open... > Run" then the following error message is created: "[19:11:19] 'Load document: 'Russula exalbicans B9-18461_1F-ITS4.cap.ac..."

Can you please help me?
Forward chromatogram *ITS1F.ab1 and reverse chromogram *.ITS4.ab1 are attached.

Bernie
https://forum.ugene.net/forum/YaBB.pl?action=downloadfile;file=Russula_exalbicans_E2+18461_1F+ITS1F.ab1 (220 KB | 1 )
https://forum.ugene.net/forum/YaBB.pl?action=downloadfile;file=Russula_exalbicans_B9+18461_1F+ITS4.ab1 (173 KB | 2 )

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Jul 16th, 2019 at 1:11pm
Hello,

I'm unable to open the attached files. Could you please try to share them again or, alternatively, share the files in another way (e.g. using Google Drive)?


Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 16th, 2019 at 8:47pm
Hello,

when I try to send a dropbox link then I get an error message

Sorry, you are not allowed to post messages containing active links to websites or images before you have posted 5 normal messages. 

Bernie

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 16th, 2019 at 10:55pm
Hello,
I have sent you the chromatogram files via researchgate.net. :)
Bernie

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Jul 17th, 2019 at 2:49pm
Hello Bernie.

It appears that that there are too many mismatches between the reads. I think, this is the reason why CAP3 was not able to produce the result.

I had to make a workaround to align the reads:
* Open both *.ab1 files in UGENE.
* In the Project View at the left part of the UGENE window select "[s]" objects of the *.ab1 files. In the context menu select "Export sequences as alignment". Thus, the reads are opened in the Alignment Editor.
* Select the second sequence in the Alignment Editor and convert it to it's reverse-complement (select "Actions > Edit > Replace selected rows with reverse-complement").
* On the "Pairwise Alignment" tab of the options panel in the Alignment Editor select the both sequences, optionally configure other parameters and click "Align".

See the result alignment in the attachment.

Note that you may select different options for calculating consensus of the reads. And export the consensus sequence, if required.
https://forum.ugene.net/forum/YaBB.pl?action=downloadfile;file=Russula_exalbicans.aln (3 KB | 305 )

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 17th, 2019 at 11:21pm
Hello Olga,

thank you for the hints!
Is it possible to show both chromatograms aligned one above the other and to edit them manually to optimize the consensus sequence?  :)

Bernie

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Jul 18th, 2019 at 11:30am
Bernie, you're welcome!


Quote:
Is it possible to show both chromatograms aligned one above the other and to edit them manually to optimize the consensus sequence?

In the current UGENE version you will have to export the consensus sequence and then map the reads to it as reference ("Tools > Sanger data analysis > Map reads to reference"), tweak the input parameters, if required.

The result will be opened in the Sanger Reads Editor (https://www.youtube.com/watch?v=lDovNM1oZEw).

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 18th, 2019 at 12:04pm
When I try to do this it results in an error (please see attachment)
Bernie
Map_Sanger_reads_to_reference_-_Error.jpg (71 KB | 329 )

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Jul 18th, 2019 at 2:40pm
Which consensus sequence did you use? I've tried to export the consensus, calculated with the "Levitsky" algorithm and the reads were successfully mapped to it. See in the attached archive the consensus and the *.ugenedb file that can be opened with the Sanger Reads Editor.
https://forum.ugene.net/forum/YaBB.pl?action=downloadfile;file=Archive.zip (47 KB | 252 )

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 21st, 2019 at 4:12am
Hello Olga,

now it works well!! But it is a long and "dangerous" way to reach the Sanger reads mapping!  I have chosen the Levintsky Consensus type.

I would like to ask you two more questions:

a) When exporting consensus there is a small box "Keep gaps". I left it open. Is that correct?


b) When at the end editing the chromatograms to optimize the consensus sequence: Is it possible to search a string whithin a chromatogram? For instance the string "GGATCATTA" or "TTGACCTCAAATC".

Thank you in advance!

Bernie

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Jul 22nd, 2019 at 3:26pm

Quote:
now it works well!!

Great!


Quote:
But it is a long and "dangerous" way to reach the Sanger reads mapping!

In future we plan to open the Sanger reads de novo assembly result directly in the Sanger Reads Editor.
Maybe we should also choose some other algorithm for de novo assembly, so that reads with low similarity (like in this case) are also taken into account.


Quote:
a) When exporting consensus there is a small box "Keep gaps". I left it open. Is that correct?

It doesn't matter for this scenario. Gaps ("-") may be inserted into the reference on the mapping step, if required (i.e. if the reads contain some insertion in comparison with the reference).


Quote:
b) When at the end editing the chromatograms to optimize the consensus sequence: Is it possible to search a string whithin a chromatogram? For instance the string "GGATCATTA" or "TTGACCTCAAATC".

Sorry, but this is one more issue we need to improve. You can do the search in the Sequence View, for example, but for now you can't do it in the Sanger Reads Editor.

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Jul 22nd, 2019 at 5:48pm
Thank you once more!

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Jul 22nd, 2019 at 5:50pm
You're welcome!

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Sep 26th, 2019 at 5:21pm
Hello,

what do you think, when will the next version of UGENE be on line?

Bernie

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Sep 26th, 2019 at 5:27pm
The next version 33 will come out very soon, but it will not contain changes for Sanger reads de novo assembly.

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Bernie on Sep 27th, 2019 at 12:36pm
Hello,

thank you for the information!
To solve my problem, would it be correct to start in the following way:
"Tools > Sanger data analysis > Map reads to reference...", choose the forward chromatogram as reference and then map forward and reverse chromatogram to that reference?
This way would be a bit sinpler to go.

Bernie

Title: Re: How to create consensus sequence from forward and reverse chromatogram
Post by Olga Golosova on Sep 27th, 2019 at 12:58pm
Hello, Bernie.


Quote:
To solve my problem, would it be correct to start in the following way:
"Tools > Sanger data analysis > Map reads to reference...", choose the forward chromatogram as reference and then map forward and reverse chromatogram to that reference?
This way would be a bit sinpler to go.

Yes, this approach can also work, if the reads intersection (i.e. the aligned region) is long enough.

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