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TotalSeq Analysis Methods And Results For Healthy Control Vs Uveitis Patients

This documentation demonstrates the use of workflow components to analyze two demo samples. Please note that the results presented in this documentation are limited in scope to the demo data. If you are rerunning the workflow, please rename the "Results" folder and move it to the "Archive" folder. Then, create a new "Results" folder and inside it, create the following folders - "qualitycontrol", "normalization", "clustering", "markers", "annotation", "subcluster", "trajectory".

Protocol

The experiment protocol information is available in this PDF document.

Sample Information

The sample information is available in this tab delimited text file.

Data Analysis Workflow

  1. Primary Analysis

Most of all the following workflow components use Seurat within R workflow. The .Rmd files are the main files. Any changes to the workflow components has to be made in the .Rmd files. The .Rmd files generate the .R and .RData files. To run/rerun these workflow, you can load the corresponding .R files or .Rmd files in Rstudio. If you want to edit the previous results, just load the corresponding .RData file and the .R or .Rmd file and make the edits. For example, if you want to run the quality control component, ALL OVER again, after adding or removing a sample, and simultaneously genenate the html report, you could run the below command in the R console, in a sinteractive session, cd /data/../TotalSeq/Tools module load R R rmarkdown::render("qualityControl.Rmd")

  1. Quality Control
  2. Normalization
  3. Clustering
  4. Differential Expression Analysis
  5. Annotation

Scripts

All the below scripts used for this analysis are available in the Tools folder;

  1. prep.sh : this BASH script takes a sampleinformation tab delimited file and samples name, creates samples folder with library.csv and swarm file
  2. organizeout.sh : this BASH scripts is run after cellranger swarm run, to move/organize output files to the results folder
  3. cellrangerReport.R : this R script Reads all the cellranger summary metric files for all the samples and plots the aggregated data for easy comparison and QC

Project Folder Structure/Contents

The project folder /data/../TotalSeq/ has the following subfolders and relevant contents as noted below;

  1. index.html : The main page to all project documentation
  2. Protocol : Contains experiment/protocol details
  3. RawData : Contains all the raw data obtained from the sequencing core
  4. Reports : Contains all the reports, including this documentation
  5. Results : Contains all the results
  6. SampleInformation.tab : Contains the sample information
  7. Tools : Contains all the scripts and reference files

Reference

  1. BioConductor