library type warning

[Rob]

Dear Dr. Patro,

I am using SALMON for my RNASeq project. Currently, I used SALMON to get the quantification value for my TNBC (Triple Negative Breast Cancer) RNASeq samples. 

I got an message: "Greater than 5% of the fragments disagreed with the provided library type; check the file: /runjobs/SP00011781/34781/R38-6_S30/lib_format_counts.json for details". 

Is this a very important error message and It is telling me that something is wrong with my processing.

Hope you help me to understand and resolve this issue. I greatly appreciate your valuable time and insights!

Regards,

Bikram Sahoo

[Rob]

Hi Bikram,

  I moved this post to it's own thread as,  previously, it was a response to the survey announcement.  The short answer to your question is that this is not, by itself, necessarily a serious error or problem.

This means that salmon detected that > 5% of the data disagreed with (i.e. was not compatible with) the fragment library type you provided.  For example, you said the data was paired end, but the strand

specificity was less than 95%, or you said it was unstranded and there was a strand bias > 5%.  This threshold (5%) is fairly conservative, and we decided it's better to warn the user of a potential  issue than

to let it go undiscovered.  Of course, you should take a look at the json file to see how different the rate actually is, and double-check that you are passing the right library type.

Best,

Rob

[Bikram Sahoo]

​Hi Dr. Patro,

Thanks so much for the great information. Appreciate you time.

Regards,

Bikram Sahoo​

--
Sailfish is available at https://github.com/kingsfordgroup/sailfish
Citation:
Patro, Rob, Stephen M. Mount, and Carl Kingsford. "Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms." Nature biotechnology 32.5 (2014): 462-464.
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[Bikram Sahoo]

Hi Dr. Patro,

I did not filter the low quality reads (phred < 30) and used SALMON for quantification purpose. Will it effect my analysis? I will really appreciate your suggestions and it will be a great help for my analysis.

thanking you,

Regards,

Bikram Sahoo

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