Hello

I'm currently using the HTqPCR package to analyse some serum microRNA data generated on the OpenArray platform of the QuantStudio 12K Flex Instrument.

The part I am currently having difficulty with is setting the flags (ThroughHole.Outlier data) to unreliable using the setCategory function. I have no problem using the setCategory function to highlight high/low Ct values just the flags.

> library(HTqPCR)

>  data(raw)

> head(featureCategory(raw), n =10)

                            Sample 1     Sample 2     Sample 3
                        Undetermined Undetermined Undetermined
002331_hsa-miR-409-5p_A Undetermined Undetermined Undetermined
000604_hsa-miR-424_A    Undetermined Undetermined Undetermined
000602_hsa-miR-30b_A              OK           OK           OK
002112_hsa-miR-29a_A    Undetermined           OK           OK
001277_hsa-miR-485-3p_A           OK           OK Undetermined
001821_hsa-miR-484_A              OK           OK           OK
000569_hsa-miR-380-3p_A Undetermined Undetermined Undetermined
002318_hsa-miR-453_A    Undetermined Undetermined Undetermined
001036_hsa-miR-485-5p_A Undetermined Undetermined Undetermined

> raw.cat <- setCategory(raw, groups=NULL, Ct.max = 35, Ct.min = 10)​

Categories after Ct.max and Ct.min filtering:
             Sample 1 Sample 2 Sample 3
OK                234      256      286
Undetermined      653      621      593
Unreliable          9       19       17

> head(flag(raw), n=10)
                        Sample 1 Sample 2 Sample 3
                          Failed   Failed   Failed
002331_hsa-miR-409-5p_A   Failed   Failed   Failed
000604_hsa-miR-424_A      Failed   Failed   Failed
000602_hsa-miR-30b_A        Pass     Pass     Pass
002112_hsa-miR-29a_A      Failed     Pass     Pass
001277_hsa-miR-485-3p_A   Failed     Pass   Failed
001821_hsa-miR-484_A        Pass     Pass     Pass
000569_hsa-miR-380-3p_A     Pass   Failed   Failed
002318_hsa-miR-453_A      Failed   Failed   Failed
001036_hsa-miR-485-5p_A   Failed   Failed   Failed

> raw.cat <- setCategory(raw.cat, groups = NULL, flag = TRUE, flag.out = "Failed")

Categories after Ct.max and Ct.min filtering:
             Sample 1 Sample 2 Sample 3
OK                234      256      286
Undetermined      653      621      593
Unreliable          9       19       17

For some reason that I can't for the life of me figure out why, is that the flags are not been marked as unreliable and was hoping someone could help me figure out where I am going wrong.

Hope this makes sense.

Thanks
Sam

Sam,

I believe this is the same problem I ran into recently:

Possible bug in setCategory function from HTqPCR package

There appears to be a bug in the setCategory function, but I still haven't received a response from the package maintainer.

Best,

Matt

Ah okay, Thanks!

I will keep an eye out on your post as well then.

Thanks

Sam