Signalprocessing: Key mixture GACT/TCAG

[sklages]

Hi,

on one region of a run we have mixed an amplicon and a shotgun
library for testing purposes.

The CWF file tells me, that there are

GACT = 71053 sequences
TCAG = 70200 sequences

Signalprocessing votes for the majority and calls bases for the GACT key
containing sequences.

What is the easiest way to get the sequences with the TCAG key?

thanks, Sven

[flxlex]

First, there is the obvious warning in the manual:

DO NOT mix samples prepared with the Rapid Library protocol with ones prepared with the FLX Standard protocol in the SAME region of the PTP device. The software cannot process data which contains different keys in the same region.

I found something that might work. You may be familiar with using custom filter parameters for processing. You start by running

gsRunProcessor --template=filterOnly > myShotgunTemplate.xml

The manual states that, although not recommended , it is possible to turn off the Key Pass filter entirely by adding the following line under the <qualityFilter> section of the template xml file:

<doClassifierCheck>false</doClassifierCheck>

Then, run the analysis again:

runAnalysisFilter --pipe=/Path/to/myShotgunTemplate.xml /Path/to/D_folder

I'm guessing you will get both your keys output, but also non-key containing reads. You might need to check the sfffile (using sffinfo) for the actual key sequence for each read.

Let me know if this works!

[sklages]

I know that I shouldn't mix both library types ... but as all keys are counted and registered in
the CWF files, there must be a way to force key detection to use a given sequence, not the majority count.

Skipping the the Key Pass Filter is, hmm, .. straight foward. Haven't thought about that :-)

Extracting the sequences according to their key then, shouldn't be much of a problem ..

I will give it a try to see at least if it works with "doClassifierCheck" flag.

thanks,
Sven

[sklages]

Hmm, after applying "doClassifierCheck" and subsequent refiltering, I get ~56.000 sequences, all with 'GACT' key .. no other "keys" :-(

That's even less what gsRunReporter is reporting on the original signal processing,
GACT = 71053 sequences
TCAG = 70200 sequences

And history tells me that this flag has been used: <doClassifierCheck>false</doClassifierCheck>

Seems not to be that easy :-) (or I have missed something very fundamental)

[RCJK]

Maybe you could try some variation on what is written below. I had to do this a couple of years ago when the Rapid Libraries were new and the key sequence wasn't recognised at first.

Look for the following file in the R_drirectory
dataRunParams.parse


Towards the end of the file you should see the following statement:

sequencesToSearchFor
{
useKitDefaults = true;
}

you will need to delete those three lines and add:


sequencesToSearchFor
{
testFragments = *,"AVTF";
libraryKey = *,"GACT";
}

Once you have done this, you will have to re-run the analysis (Using the runAnalysisPipe command in a teminal window)

[sklages]

yes, it's autoDetectKeys in current versions.

But processing remains fairly unimpressed by the changes made:

Key Sequence: GACT

It seems that manual interaction is simply ignored ..

[HMorrison]

no easy solution

We did this by mistake a while ago (March 2011)--after trying various ways to get the information ourselves, had to send the run files to GS Support where they were able to get the two datasets extracted for us.