TRaceR

Pipeline

This is a Kerblam! data analysis project. Provided that you have the right input data structure, just run the tracer :: Multiparametric Calcium Trace Analyzer workflow through

kerblam run tracer

to get the final output.

A run-time log will be printed on screen, but it is not saved anywhere by default. Run

kerblam run tracer 2>&1 | tee ./data/out/log.txt

to save it locally.

You can also run stat_tracer :: TRaceR - Statistical Analysis Only if you want to run only the statistical analysis step, following the first step of time course analysis.

Requisites

R packages

• r4tcpl
• stats
• dplyr
• tidyr
• ggplot2
• patchwork
• zoo
• pracma

Kerblam! (>= 1.2.1)

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Biological Model

Primary cell cultures of mouse cerebellar granules for the study of pharmacologically-induced ataxia-telangiectasia.

ATM (Ataxia-telangiectasia mutated) is a crucial serine/threonine protein kinase activated primarily by DNA double-strand breaks and oxidative stress. As a central regulator of the DNA damage response (DDR), ATM phosphorylates substrates like p53, BRCA1, and Nbs1 to maintain genomic stability. Beyond DNA damage, ATM functions as a sensor for oxidative stress, managing reactive oxygen species (ROS) levels. Deficiencies in ATM cause Ataxia-telangiectasia, characterized by neurodegeneration.

Biological Readout

The response of cerebellar granules to UV stimulation for the induction of ROS production was recorded by sampling (1 Hz) their cytosolic calcium concentration using the non-ratiometric, green-emitting FLUO-4 probe, which features a maximum excitation at 488 nm (since FURA-2, which is excited in the UV range, would have interfered with the treatment).

Data matrices from these fluorescence measurements are used as the input data of TRaceR. Each one of them corresponds to an independent biological observation of intracellular calcium dynamics, performed under some specific experimental condition. Within such matrices, each column represents from a different Region Of Interest (ROI), corresponding to a single-cell trace.

Experimental Conditions

• DMSO: control condition (KU vehicle - always present)
• UV: inducer of ROS production (always present)
• KU: ATM protein inhibitor (-> ataxia-telangiectasia mimicker)
• NAC: N-acetylcysteine (antioxidant agent)
• OMO: activator of NRF2 (a ROS-protective gene expression program)
• CBX: carbenoxolone (gap junction and pannexin-1 blocker)
• Gd: gadolinium (calcium channel blocker)
• RR: Ruthenium Red (inhibitor of TRP channels, TRPV in particular)
• DFO: deferoxamine (iron chelator)
• BAPTA: selective calcium chelator
• TROLOX: analogue of Vitamin E (antioxidant agent)
• U73122: phospholipase C (PLC) inhibitor

1. Batch KU_Gd_CBX
   • KU
   • KU_CBX
   • KU_Gd
   • KU_Gd_CBX
2. Batch KU_lessUV_NAC
   • DMSO
   • DMSO_NAC
   • KU
   • KU_lessUV
   • KU_NAC
3. Batch KU_OMO
   • DMSO_OMO
   • KU
   • KU_OMO_24h
   • KU_OMO_48h
4. Batch KU_RR_DFO_BAPTA
   • KU
   • KU_BAPTA
   • KU_DFO
   • KU_RR
5. Batch KU_TROL500 (500 µM 1h-preincubation)
   • KU
   • KU_TROL500
   • KU_TROL500_30m
6. Batch KU_TROLOX (100 µM O/N)
   • KU
   • KU_TROLOX
7. Batch KU_U7
   • KU
   • KU_U7

Data Format

The input data must be organized as follows: for each batch (KU_OMO, KU_lessUV_NAC, etc.), there must be a folder containing a subfolder for each condition (e.g., DMSO, KU, KU_NAC, etc.). Since the names of these folders will be used to identify batches, groups, and perform comparisons, avoid using spaces in the folder names.

It is assumed that each input file—representing a biological replicate—consists of a CSV organized as follows:

• , for decimal point
• ; as field separator
• a string in the first row to skip
• column headings in the second row
• fileEncoding UTF-16LE (with BOM)

Finally, each experiment is expected to last 900 seconds, at a sampling rate of 1 second. Otherwise, the statistics may no longer be comparable.

Statistical Contrasts

Contrasts need to be defined inside the input folder of each batch through a plain text file, named comp.txt (or comparisons.txt), where each statistical comparison is listed in the form

condition -- reference

Blank lines allowed, as well as comment lines introduced by #.

Anomalies

Recording Length

The standard duration of each experiment should be exactly 900 seconds, at a sampling rate of 1 second. The following files are exceptions.

Only 600 time samples for ./data/in/KU_lessUV_NAC/DMSO experiments

• 250908_011_DMSO.csv
• 250908_012_DMSO.csv
• 250908_013_DMSO.csv

Only 700 time samples for ./data/in/KU_lessUV_NAC/KU_NAC experiments

• 250908_045_KU_NAC.csv

However, given the particular experimental condition they belong to (eliciting just small or no responses), we believe they do not introduce statistical artifacts.

In fact, recordings from ./data/in/KU_TROL500/KU_TROL500_30m

• 260329b_510_KU_TROL500_30m.csv
• 260329b_511_KU_TROL500_30m.csv
• 260329b_610_KU_TROL500_30m.csv
• 260329b_611_KU_TROL500_30m.csv

are the only ones that cannot be compared with the others, featuring large-amplitude calcium signals, but being twice as long as the standard duration (30 minutes vs 900 seconds).

CSV separators

./data/in/KU_OMO/KU/251113_011_KU.csv was the only CSV input file with a different separator convention, even though it used the same file encoding, UTF-16LE (with BOM). This caused the analysis in the ./data/in/KU_OMO/KU/ folder to fail, although it did not stop the entire program from running. It was fixed by:

fpath <- "./data/in/KU_OMO/KU/251113_011_KU.csv"
fpath |> read.table(
        header = TRUE,
        sep = "\t",
        dec = ".",
        skip = 1,
        check.names = FALSE,
        stringsAsFactors = FALSE,
        fileEncoding = "UTF-16LE") |>
    lapply(as_num) |> as.data.frame() |> write.table(
        file = "./data/in/KU_OMO/KU/251113_011_KU_new.csv",
        sep = ";",
        dec = ",",
        row.names = FALSE,
        col.names = TRUE,
        quote = FALSE,
        fileEncoding = "UTF-16LE")

Then the first-line string Channel.002\r\n was manually reinserted in ./data/in/KU_OMO/KU/251113_011_KU_new.csv and saved as ./data/in/KU_OMO/KU/251113_011_KU.csv.