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Re: [Biojava-l] Aligning Chromatogram with Phrap ACE sequences ? Ashika Umanga Umagiliya Wed Jun 17 03:00:20 2009

Hi again Andy ,

Thank you for all your help , it was a matter of setting an option in ChromatogramGraphic


ChromatogramGraphic chromaGfx;
.
.
chromaGfx.setOption(Option.SUBPATH_LENGTH, new Integer(
                   Integer.MAX_VALUE));


Now I can see the entire trace-plot ..  Everything is just fine :)


Best Regards,
Umanga

Andy Yates wrote:
Hi Umanga,

Glad you've got it rendering though now :)

My best guess is that the trace have been truncated by the generating
program or the file writing is incomplete which I doubt due to the way
SCF files are constructed. So my money is on Phrap/Phred doing a bit of
quality control with the end of the trace. Why it hasn't truncated the
base calls as well I do not know and is probably a question better
suited to the people who write/maintain Phred & Phrap.

Sorry I can't be of that much more help :(

Andy

Ashika Umanga Umagiliya wrote:
Hi Andy ,

Thanks for the help.I was doing all wrong !
Instead of using AB1 raw data , I used SCF  generated by Phrep and used
the offset values from ACE file.
Now all are aligned , here a snapshot : http://i39.tinypic.com/xbymf.png

But still one problem .As can be seen in the picture, at the end of the
graph, some part of the trace-plot is missing for last bases.
Any idea whats going on here ?

Thanks in advance,
Umanga

Andy Yates wrote:
Hi Umanga,

In terms of getting the offset I'm not sure. That information should be
provided via the ACE file or from some other output from phrap; its not
the responsibility of the AB1 file to contain this information. The
phred output (which phrap uses under the scenes I think) which does
contain this information is the SCF output but in this situation you are
not rendering the processed output from the ABI sequencer. You are
actually displaying:

ABI Raw output -> phred basecalled/processed

So if there is a reason why you want to show the ABI processed data then
I'm not sure how to marry it to the phrap output. Otherwise just use the
traces phrap outputs and see what happens.

Andy

Ashika Umanga Umagiliya wrote:
Hi Andy,

Thank you for the reply.
Earlier problem was I had not applied the compliment in the
ChromatoGraph.
Now I could inspect the alignment with the Phrap calls

Please refer :  http://i43.tinypic.com/e5iu12.png

I have drawn orange underlines from where the two sequences matches.
But I had to hardcode x1 and x2 offset values to make the graphs align
with the Phrap basecalls.
So as for my question
http://lists.open-bio.org/pipermail/biojava-l/2009-June/006707.html , if
somehow get all (or fill) all the callbases same as in ACE , I can align
easily.


Best Regards,
Umanga





Andy Yates wrote:
Hi Ashika,

AB1 files will contain a different basecall to one generated by phrap
since (as far as my memory serves me) ABI's platform has a basecaller
built in. This is the one which is coded into the AB1 file & therefore
chromatogram graphic will pick up on that one. However it's very odd
that the Phrap calls are so distant from the trace itself.

I would have hoped that the ACE file would have contained some kind of
called base -> peak position in the chromatogram. There is always the
option of getting phred to output a SCF file which should contain this
information & see what it thinks its doing. SCFs can be parsed by
BioJava so you should be able to push it straight into your program &
let it handle it.

Andy

Ashika Umanga Umagiliya wrote:
Greetings all,

Please refer to image 1 : http://i40.tinypic.com/11rsk00.png
image 2 (end of graph):  http://i44.tinypic.com/2vt4iu8.png


As shown in image (1) , I draw the basecall sequences of AB1 file,by
parsing the ACE file generated by Phrap.That is ,for upper part I
do not
read AB1 file.Only read values from ACE file.(values under 'RD' tag in
ACE file.)In the example, two files are 'sampleA-S-R.AB1' and
'sampleA-S-F.AB1'.

For the bottom part- that is to draw Chromatogram, I use Biojavas
'ChromatogramGraphic' class.

I wanted to align these two parts- that is align 'sampleA-S-R.AB1'
basecall sequence with 'sampleA-S-R.AB1' chromatogram..and so
on...(like
in ChromasPro).

But I noticed that basecall sequence of 'ChromatogramGraphic' is
totally
different than that of from ACE file.
Refer to image (2) ; you can see the two sequences generated by
same AB1
file is different in lenght also.

What could be the problem here? Any suggestions please

thanks in advance.
umanga
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