J1994.scRNA-TCR

This repository contains processed data and analysis scripts used in the manuscript by Huff et. al, "An off-the-shelf vaccine activates a broad repertoire of mutant KRAS-specific T cells in patients with resected pancreatic cancer". Code written by Alexander Girgis and Ludmila Danilova.

Directory Structure

R code files are located in parent directory. These files should be executed in their current location relative to all subfolders in directory.

/codeoutputs/: Empty folder which will become populated with resluts files upon executing R code files. /rfunctions/: Code folder contaning miscellaneous helper functions in processing TRBV nomenclature. /rawdata/: Contains scRNA and scVDJ output from CellRanger pipeline. Currently not available due to data sharing permissions, but will be uploaded to dbGaP and appropriate repositories. /codeinputs/: Processed data objects loaded and used in at least one of the R code files. Currently not available due to data sharing permissions, but will be uploaded to dbGaP and appropriate repositories.

Scope of Analysis Scripts

The contents of this repo include: 1. QC and annotation of single-cell RNA data for 5 subjects receiving mKRAS-Vax. Isolation of annotated T cells from single-cell data. - A.20230529Read10x.r: Process CellRanger output into aggregate Seurat object. - B.20230606sc-by-batch.r: Split aggregate object by sequencing batch. Perform separate QC and filtering. - C.20231108sc-cluster-assignment-batch1.r: Annotate putative T cell clusters based on cluster markers and Azimuth annotation for sequencing batch 1. - D.20231108sc-cluster-assignment-batch2.r: Annotate putative T cell clusters based on cluster markers and Azimuth annotation for sequencing batch 2. 2. Analysis of T-cell expansion assays to identify clonotypes expanded to mKRAS peptides. - E.20230505DiffExFESTData.r: Analyze expansion assay data to identify T cell receptor clonotypes expanded to mKRAS peptides. - F.20240418MakeFilteredDiffExTable.r: Generate compiled table of all mKRAS expanded TCRs 3. Integration of single-cell and TCR expansion data to identify anti-mKRAS T cells in single-cell data. - G.20240417scKRASAnnot.r: Integrate mKRAS expansion data and single-cell data to create a new Seurat object with annotations for anti-mKRAS T cell reactivity. - H.20240508pullAllmKRASwithExpansionInfo-all.r: Generate composite table containing paired chain information, T cell phenotype, and mKRAS reactivity for anti-mKRAS TCRs in scRNA data. - I.20240427MinorEditingObjects.r: Combine annotated T cell objects for batch 1 and batch2 back into one seurat object for convenience. 4. Analysis of gene expression profile of mKRAS-specific T-cells. - J.2024-04-23Seuratviz.Rmd 5. Comparison of mKRAS TCRs with TCGA data. - K.024-04-29_TCGA.Rmd

Other Files

    LICENSE: GNU v3.0 license governing the use and sharing of code in this repository. 
    SESSIONINFO: R Package versions for libraries used in analysis scripts for I-III described above.