Ladder and bp sizing

Find the ladder peaks in and use that to call bp size

Usage

find_ladders(
  fragments_trace,
  ladder_channel = "DATA.105",
  signal_channel = "DATA.1",
  ladder_sizes = c(50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490,
    500),
  ladder_start_scan = NULL,
  minimum_peak_signal = NULL,
  scan_subset = NULL,
  ladder_selection_window = 5,
  max_combinations = 2500000,
  warning_rsq_threshold = 0.998,
  show_progress_bar = TRUE
)

Arguments

fragments_trace

list from 'read_fsa' function

ladder_channel

string: which channel in the fsa file contains the ladder signal

signal_channel

string: which channel in the fsa file contains the data signal

ladder_sizes

numeric vector: bp sizes of ladder used in fragment analysis. defaults to GeneScan™ 500 LIZ™

ladder_start_scan

numeric: indicate the scan number to start looking for ladder peaks. Usually this can be automatically found (when set to NULL) since there's a big spike right at the start. However, if your ladder peaks are taller than the big spike, you will need to set this starting scan number manually.

minimum_peak_signal

numeric: minimum signal of peak from smoothed signal.

scan_subset

numeric vector (length 2): filter the ladder and data signal between the selected scans (eg scan_subset = c(3000, 5000)). to pracma::savgol().

ladder_selection_window

numeric: in the ladder assigning algorithm, the we iterate through the scans in blocks and test their linear fit ( We can assume that the ladder is linear over a short distance) This value defines how large that block of peaks should be.

max_combinations

numeric: what is the maximum number of ladder combinations that should be tested

warning_rsq_threshold

The value for which this function will warn you when parts of the ladder have R-squared values below the specified threshold.

show_progress_bar

show progress bar

Value

This function modifies list of fragments_trace objects in place with the ladder assigned and base pair calculated.

Details

This function takes a list of fragments_trace files (the output from read_fsa) and identifies the ladders in the ladder channel which is used to call the bp size. The output is a list of fragments_traces.

In this package, base pair (bp) sizes are assigned using a generalized additive model (GAM) with cubic regression splines. The model is fit to known ladder fragment sizes and their corresponding scan positions, capturing the relationship between scan number and bp size. Once trained, the model predicts bp sizes for all scans by interpolating between the known ladder points. This approach provides a flexible and accurate assignment of bp sizes, accommodating the slightly non-linear relationship.

Use plot_data_channels() to plot the raw data on the fsa file to identify which channel the ladder and data are in.

The ladder peaks are assigned from largest to smallest. I would recommend excluding size standard peaks less than 50 bp (eg size standard 35 bp).

Each ladder should be manually inspected to make sure that is has been correctly assigned.

See also

plot_data_channels() to plot the raw data in all channels. plot_ladders() to plot the assigned ladder peaks onto the raw ladder signal. fix_ladders_interactive() to fix ladders with incorrectly assigned peaks.

Examples

fsa_list <- lapply(cell_line_fsa_list[1], function(x) x$clone())

find_ladders(fsa_list, show_progress_bar = FALSE)

# Manually inspect the ladders
plot_ladders(fsa_list[1])