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Re: [Biojava-l] Aligning Chromatogram with Phrap ACE sequences ? Andy Yates Wed Jun 17 02:00:34 2009

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
>>>>> _______________________________________________
>>>>> Biojava-l mailing list  -  [EMAIL PROTECTED]
>>>>> http://lists.open-bio.org/mailman/listinfo/biojava-l
>>>>>             
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