Question

Technical Replicates in HTqPCR

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Ed Siefker ▴ 230

@ed-siefker-5136

Last seen 5 months ago

United States

How does HTqPCR handle technical replicates?

Suppose I have 4 samples, 8 primer sets, and 3 replicates on a 96 well plate.

"n.features indicates the number of features present on each array."

Features are genes? So 8.

"n.data...Indicates the number of samples that are present in each file."

Samples are 4

"n.features*n.data must correspond to the total number of lines to be read from each file. "

8*4=32 But I have 96 lines of data?

Do I have to make each technical replicate it's own feature? How then would I indicate that they are replicates if they each have their own feature name? I can't reuse feature names because I can't reuse column names.

What am I not understanding?

HTqPCR • 385 views

ADD COMMENT • link written 7 months ago by Ed Siefker ▴ 230

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My actual data set has 12 samples, 8 genes, and 3 technical replicates. I'm trying with n.features=24 and it's not working.

Here's what my file looks like:

> head(read.csv(files))
      position indiv_PCR_eff  feature        Sample.Name threshold mean_PCR_eff     Ct
1    AA1_Best1         1.000    Best1 Farnesol KO noTX 3     0.228        1.936  0.000
2     AA2_PKLR         2.004     PKLR Farnesol KO noTX 3     0.218        1.957 34.118
3     AA3_PLCB         1.000     PLCB Farnesol KO noTX 3     0.307        1.879  0.000
4 AA4_Phospho1         1.000 Phospho1 Farnesol KO noTX 3     0.124        1.861  0.000
5    AA5_GREM2         1.000    GREM2 Farnesol KO noTX 3     0.130        1.891  0.000
6    AA6_PLIN4         2.006    PLIN4 Farnesol KO noTX 3     0.350        1.920 36.216

column.info is set up correctly, matching the column numbers in the data file

> class(column.info)
[1] "list"
> column.info
$feature
[1] 3

$position
[1] 1

$Ct
[1] 7

samples is populated with the Sample.Name column from the data file

> samples <- read.csv(files)[["Sample.Name"]]
> class(samples)
[1] "character"

But the call to readCtData() doesn't work.

> readCtData(files,path=".", n.features=24, n.data=12, format="plain", header=TRUE, samples=samples, column.info=column.info)
Error in `[.data.frame`(sample, , column.info[["Ct"]]) : 
  undefined columns selected

It looks like I've done everything according to the documentation. What am I missing?

ADD REPLY • link 7 months ago Ed Siefker ▴ 230

score 0 · Answer 1 · 2023-09-19

I've never used HTqPCR, so take this with a grain of salt. It appears that each sample is read in using HTqPCR:::.readCtPlain, and then the step that is failing is not finding the 'Ct' column. My usual course of action would be to inspect the input that you get from that function:

## note that there are three colons and a dot between the package and function name!
z <- HTqPCR:::.readCtPlain(samples[1], header = TRUE, n.features = 24, n.data = 12, i = 1)

And then inspect z. You could compare it to what you get if you simply do

zz <- read.delim(samples[1], header = TRUE)

That should provide some information that may be helpful to figure out what the problem is.