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De novo assembly with multiple sequences (CAP3) (Read 174 times)
May 21st, 2019 at 8:47pm

Nikolas   Offline
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Hello,

I have four reads from sanger sequencing and would like to align them with CAP3. Two of these four reads are generated with the same primer (forward), the other two with another (reverse) primer. Each primer was used twice on (theoretically) identical templates, hence four reads.

So I select Tools -> Sanger data analysis -> Reads de novo assembly (with CAP3)...
In the dialogue, I add all four .ab1 files and hit Run. The task finishes without error.

But the alignment then only shows two reads (the first two from the list). Moreover, if I instead only add the two sequences from the forward sequencing or the two from the reverse sequencing, the task aborts with the following error messages:

[ERROR][15:17] Subtask {Load document: 'PDS_1F.cap.ace'} is failed: File doesn't contain any msa objects
[ERROR][15:17] Task {CAP3 run and open result task} finished with error: Subtask {Loading documents} is failed: Subtask {Opening view for document: PDS_1F.cap.ace} is failed: Subtask {Load document: 'PDS_1F.cap.ace'} is failed: File doesn't contain any msa objects

Only read pairs with opposite direction seem to work. As far as I understand CAP3 (from reading not more than the abstract of the paper), it should be able to align any number of sequnces.

I can align the sequences fine with MUSCLE (inside UGENE) using a FASTA as input, but then I lose the quality info from the chromatograms.

Am I doing something wrong? Any help would be appreciated.

Thanks in advance,
Nikolas

MacOS 10.14.4
UGENE 1.32.0 (64 bit)
 
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Reply #1 - May 22nd, 2019 at 12:30pm

Olga Golosova   Offline
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Hello Nikolas,

Could you please send us the reads, we'll try to investigate the problem.

Quote:
As far as I understand CAP3 (from reading not more than the abstract of the paper), it should be able to align any number of sequnces.

In, general, yes, however, this doesn't work with these exact reads.You might try to tweak the CAP3 parameters.

Quote:
I can align the sequences fine with MUSCLE (inside UGENE) using a FASTA as input, but then I lose the quality info from the chromatograms.

As I understand, you have a contig sequence that you can use as a reference sequence. Try to use the second item in the menu, i.e. "Tools > Sanger data analysis > Map reads to reference".
 
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Reply #2 - May 22nd, 2019 at 5:16pm

Nikolas   Offline
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Hello Olga,

thanks for your reply.

I have attached three of the read files to this post, the fourth I will attatch to the next post, as it is only possible to attatch three 250 KB files and each of them is 150 KB.

I did not have a reference sequence and my idea was to assemble the reads without one. Now I of course can use the consensus form the MUSCLE alignment as a reference for the reads. I have tried it as a workaraound and it works fine.

It would still be more convenient to directly assemble the reads with CAP3. Which parameters would I have to tweak? As far as I can see, two  of my sequences are just being ignored and I don't see any parameters in the Advanced tab that would influence this behavior. Except maybe for the "Assembly reverse reads" flag which I have set on and off without any effect on the outcome.

Cheers,
Nikolas
 

PDS_1F_ab1.zip (148 KB | 9 )
PDS_1R_ab1.zip (145 KB | 8 )
PDS_2F_ab1.zip (146 KB | 8 )
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Reply #3 - May 22nd, 2019 at 5:17pm

Nikolas   Offline
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Read number four.
 

PDS_2R_ab1.zip (146 KB | 8 )
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Reply #4 - May 22nd, 2019 at 7:45pm

Olga Golosova   Offline
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The reason the reads are not assembled with CAP3 are different poor-quality ends of the reads. If you, for example, modify the "Clipping for poop regions" parameters of the CAP3 tool as follows:
  • "Base quality cutoff for clipping (-c)" = "30"
  • "Clipping range" = "500"

then all four reads will be present in the alignment.

However, to be able to work with the chromatograms, in the current UGENE version you'll have to map the reads to the contig sequence anyway (i.e. use CAP3, export the contig sequence from the alignment, map reads to the contig sequence).

 

Contig1.fa (1 KB | 7 )
CAP3_settings.png (67 KB | 8 )
CAP3_settings.png
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Reply #5 - May 24th, 2019 at 2:32am

Nikolas   Offline
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This worked, thank you!

I would have expected an error message of some sort, stating that not all of my reads could be aligned.

Cheers and thanks again,
Nikolas
 
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Reply #6 - May 24th, 2019 at 1:34pm

Olga Golosova   Offline
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Great! Thanks for informing me about the result!
 
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