Remove baseline from qPCR amplification curves by subtracting median of initial cycles.
Arguments
- plateamp
data frame with plate amplification data, including variables well, cycle, fluor_raw (raw fluorescence value), and program_no. Assume program 2 for amplification curves from Roche Lightcycler format data.
- maxcycle
maximum cycle value to use for baseline, before amplification.
Value
platemap with additional columns per well:
| fluor_base | baseline /background value |
| fluor_signal | normalized fluorescence signal, i.e. fluor_raw - fluor_base |
Details
BETA function version because:
- assumes Roche Lightcycler format, we should ideally replace "program_no == 2" by something more generic?
- the rule-of thumb "baseline is median of initial 10 cycles" has not been tested robustly
Examples
# create simple dataset of raw fluorescence
# with two samples over 15 cycles
raw_fluor_tibble <- tibble(sample_id = rep(c("S1", "S2"), each = 15),
target_id = "T1",
well_row = "A",
well_col = rep(c(1, 2), each = 15),
well = rep(c("A1", "A2"), each = 15),
cycle = rep(1:15,2),
fluor_raw = c(1:15, 6:20),
program_no = 2)
# remove base fluorescence from dataset
raw_fluor_tibble %>%
debaseline()
#> # A tibble: 30 × 10
#> sample_id target_id well_row well_col well cycle fluor_raw program_no
#> <chr> <chr> <chr> <dbl> <chr> <int> <int> <dbl>
#> 1 S1 T1 A 1 A1 1 1 2
#> 2 S1 T1 A 1 A1 2 2 2
#> 3 S1 T1 A 1 A1 3 3 2
#> 4 S1 T1 A 1 A1 4 4 2
#> 5 S1 T1 A 1 A1 5 5 2
#> 6 S1 T1 A 1 A1 6 6 2
#> 7 S1 T1 A 1 A1 7 7 2
#> 8 S1 T1 A 1 A1 8 8 2
#> 9 S1 T1 A 1 A1 9 9 2
#> 10 S1 T1 A 1 A1 10 10 2
#> # ℹ 20 more rows
#> # ℹ 2 more variables: fluor_base <dbl>, fluor_signal <dbl>