Processing images

Introduction

All scripts were run from the directory

cd /project2/gilad/joycehsiao/fucci-seq/code/

and under the conda environment fucci-seq. To activate the conda environment:

cd /project2/gilad/joycehsiao/fucci-seq
source activate fucci-seq

The raw image files are located at:

cd /project2/gilad/fucci-seq/code/images_curated

There are three majar steps in image processing. In each step, we use R script to perform the analysis and then use bash script to submit jobs to the cluster.

Step 1: Detect nucleus outline

Step 2: Combine images from all channels

Step 3: Compute summary metrics for image intensities

Detect nucleus outline

fucciMask.R processes data for one plate at a time, from reading in images from all four channels (/project2/gilad/fucci-seq/images_curated/plateID), detecting nucleus edge using DAPI channel, outputing cropped images fro each channel (/scratch/midway2/joycehsiao/fucci-seq/images_processed), to saving matrices identifying areas inside nucleus (/project2/gilad/fucci-seq/intensities/plateID/*.ints.rds), matrices containing pixel intensities for each channel (/project2/gilad/fucci-seq/intensities/plateID/*.ints.rds) and matrices containg the number of nuclei detected for each sample (project2/gilad/fucci-seq/intensities/*.rds).

submit-fucciMask.sh submits batch jobs running fucciMask.R for one plate a time.

bash submit-fucciMask.sh

Combine images from all channels

makeWellIdImages.R creates labels (00001, …, 00096) for the images files and saves the labels in /project2/gilad/fucci-seq/images_wellid.

fucciMask.combineImages.R combines the image labels (/project2/gilad/fucci-seq/images_wellid) with the sample cropped images (/scratch/midway2/joycehsiao/fucci-seq/images_processed) for one plate at a time, making a combined image for each single cell sample for visualization (/project2/gilad/fucci-seq/images_combined).

submit-fucciMask.combineImages.sh submits batch jobs running fucciMask.combineImages.R for one plate at a time.

bash submit-fucciMask.combineImages.sh

Compute summary metrics for image intensities

intensity.summarize.R uses R packakge EBImage to compute intensity summary statistics for the single cell samples detected to have single nucleus. The computation is done for one plate a time. Results are stored in /project2/gilad/joycehsiao/fucci-seq/intensities_stats.

submit-intensity.summarize.sh submits batch jobs running intensity.summarize.R for one plate at a time.

bash submit-intensity.summarize.sh