[BioC] AgiMicroRna -1 filtered values

[Coonen Maarten (TGX)]

Dear Steve,

> I think I'm somewhat confused ... isn't the scenario you are describing -- a miRNA is not detected in the control arrays, but detected in the (presumably the "treatment" arrays) -- the definition of differential expression?

I see your point, but this is only when one can be 100% sure that -1 means that the miRNA is not above the detection limit. In this case, the miRNA actually is low expressed and I agree with your statement above. 
But what if the probe replicates of a miRNA are not accurately measured and Feature Extraction Software (FES) decides to flag this miRNA as not-detected (gIsGeneDetected). AgiMicroRna then converts the expression for this miRNA into -1 and the miRNA continues in the analysis, while it actually did not pass the FES-QC.

On the other hand, I am in doubt if I might be misinterpreting the FES-output. Which of the options below is true? I couldn't find any of this in the FES manual.
a) FES performs QC on the probe replicates and summarizes all good probes into 1 TotalGeneSignal. Since a miRNA is represented by 30 probes, FES will always succeed in summarizing the good probes and will always generate a reliable TotalGeneSignal.
b) FES performs QC on the probe replicates. If all probes fail to pass QC, it is unable to generate a reliable TotalGeneSignal and puts a flag in the IsGeneDetected column.

If a) holds true, I agree on using the miRNAs with -1 in further analysis. 
If b) holds true, we are not able to distinguish between miRNAs that are absent and miRNAs that are not accurately measured.

> Also, have you checked to see if this scenario you are imagining is actually happening?
In the sample data set it occurs multiple times, of which hsa-miR-339-5p is the first example. 
In my own data, I have seen these -1 values occurring multiple times, which made me curious to what was causing this. 

Best regards,
Maarten

-----Original Message-----
From: mailinglist.honeypot at gmail.com [mailto:mailinglist.honeypot at gmail.com] On Behalf Of Steve Lianoglou
Sent: dinsdag 11 februari 2014 15:27
To: Coonen Maarten (TGX)
Cc: bioconductor at r-project.org; plopez at cnic.es
Subject: Re: [BioC] AgiMicroRna -1 filtered values

Hi,

On Tue, Feb 11, 2014 at 12:04 AM, Coonen Maarten (TGX) <m.coonen at maastrichtuniversity.nl> wrote:
> Dear mailing list,
>
> I'm using the AgiMicroRna-package to analyze my Agilent microRNA arrays.
> It occurred to me that the ProcessedData.txt output file contains -1 values for miRNAs that were below the 0.5 intensity threshold in the 'tgsMicroRna' function, when half=TRUE.
>
> These -1 values are subsequently used in the analysis of differential genes (limma), which can (in my opinion) lead to false positive results.
> E.g. imagine a situation where the intensities for a miRNA of 2 control samples were below the detection limit of the scanner (noise) and therefore end up with a value of -1.
> For the 2 treated samples however the intensities were higher (1.395 and 1.147 respectively).
> This would generate a logFC of 2.271 and most likely a significant p-value. As a result, this miRNA would be selected as differentially expressed, whilst actually not being measured in 2 of the 4 samples.

I think I'm somewhat confused ... isn't the scenario you are describing -- a miRNA is not detected in the control arrays, but detected in the (presumably the "treatment" arrays) -- the definition of differential expression?

Also, have you checked to see if this scenario you are imagining is actually happening?

-steve

--
Steve Lianoglou
Computational Biologist
Genentech