count_foci: count_foci

In mcneilllucy/synapsis: An R package to automate the analysis of double-strand break repair during meiosis

Description

Calculates coincident foci in synaptonemal complex and foci channel, per cell

Usage

count_foci(
  img_path,
  stage = "none",
  offset_px = 0.2,
  offset_factor = 2,
  brush_size = 3,
  brush_sigma = 3,
  foci_norm = 0.01,
  annotation = "off",
  channel2_string = "SYCP3",
  channel1_string = "MLH3",
  file_ext = "jpeg",
  KO_str = "--",
  WT_str = "++",
  KO_out = "-/-",
  WT_out = "+/+",
  watershed_stop = "off",
  watershed_radius = 1,
  watershed_tol = 0.05,
  crowded_foci = TRUE,
  artificial_amp_factor = 1,
  strand_amp = 2,
  min_foci = -1,
  disc_size = 51,
  modify_problematic = "off",
  disc_size_foci = 5,
  C1 = 0.02,
  C2 = 0.46,
  C_weigh_foci_number = TRUE
)

Arguments

img_path,	path containing crops to analyse
stage,	meiosis stage of interest. Currently count_foci determines this with thresholding/ object properties in the synaptonemal complex channel by previosly calling the get_pachytene function. Note that if using this option, the count_foci function requires that the input directory contains a folder called “pachytene” with the crops in it.
offset_px,	Pixel value offset used in thresholding of synaptonemal complex channel
offset_factor,	Pixel value offset used in thresholding of foci channel
brush_size,	size of brush to smooth the foci channel. Should be small to avoid erasing foci.
brush_sigma,	sigma for Gaussian smooth of foci channel. Should be small to avoid erasing foci.
foci_norm,	Mean intensity to normalise all foci channels to.
annotation,	Choice to output pipeline choices (recommended to knit)
channel2_string	String appended to the files showing the channel illuminating synaptonemal complexes. Defaults to SYCP3
channel1_string	String appended to the files showing the channel illuminating foci. Defaults to MLH3
file_ext	file extension of your images e.g. tiff jpeg or png.
KO_str	string in filename corresponding to knockout genotype. Defaults to –.
WT_str	string in filename corresponding to wildtype genotype. Defaults to ++.
KO_out	string in output csv in genotype column, for knockout. Defaults to -/-.
WT_out	string in output csv in genotype column, for knockout. Defaults to +/+.
watershed_stop	Stop default watershed method with "on"
watershed_radius	Radius (ext variable) in watershed method used in foci channel. Defaults to 1 (small)
watershed_tol	Intensity tolerance for watershed method. Defaults to 0.05.
crowded_foci	TRUE or FALSE, defaults to FALSE. Set to TRUE if you have foci > 100 or so.
artificial_amp_factor	Amplification of foci channel, for annotation only.
strand_amp	multiplication of strand channel to make masks
min_foci	minimum pixel area for a foci. Depends on your dpi etc. Defaults to 4
disc_size	size of disc for local background calculation in synaptonemal complex channel
modify_problematic	option for synapsis to try and "save" images which have likely been counted incorrectly due to a number of reasons. Default settings are optimized for mouse pachytene. Defaults to "off"
disc_size_foci	size of disc for local background calculation in foci channel
C1	Default crispness criteria = sd(foci_area)/(mean(foci_area)+1)
C2	Alternative crisp criteria.
C_weigh_foci_number	choose crispness criteria- defaults to TRUE to use C1 (weighing with number). Otherwise set to FALSE to use C2

Details

In this function, masks for the synaptonemal complex (SC) and foci channel are created from the saved crops of single/individual cells. These masks are computed using (optional) input parameters related to meiosis stage/ how well spread chromosomes are (for the former) and related to smoothing, thresholding and how "crowded" foci are for the latter. Finally, these two masks are multiplied, and the number of objects found with EBImage's computeFeatures are the colocalizing foci.

The file, cell number, foci count etc. are output as a data frame.

Value

data frame with foci count per cell

Author(s)

Lucy McNeill

Examples

demo_path = paste0(system.file("extdata",package = "synapsis"))
foci_counts <- count_foci(demo_path,offset_factor = 3, brush_size = 3,
brush_sigma = 3, annotation = "on",stage = "pachytene")

mcneilllucy/synapsis documentation built on Dec. 21, 2021, 3:59 p.m.