Zebrafish-VDJ-analysis (WIP)

Code and tutorial for the analysis of V(D)J recombination in the zebrafish, using RNA-Seq data and the MIXCR tool. Here is provided the reference sequence of the zebrafish TRB locus, obtained from Meeker et al, 2010, which, as of October 2nd, 2021 had not yet been deposited in the IMGT database.

1. Install MIXCR and add zebrafish TRB sequences

1.1. Follow the instructions of the developers as detailed here. 1.2. Download the file provided here (add hyperlink to the json library) and save it in the library folder of the downloaded MIXCR tool, as detailed here. In short, as follows:

After mixcr installation, try, in the command line:

mixcr -v

Navigate to the folder specified in the command line (e.g., /usr/local/Cellar/mixcr/3.0.13-2/libraries), and save there the external library containing the reference TRB sequences.

2. Clone vdjveR from Github

vdjveR is an R package which contains functions that may help exploring VDJ diversity after alignment with MIXCR. One of the potentially useful functions is the calculation of equitability scores for the evaluation of the equivalence of the clones within a population.

Clone vdjveR directly from the github repository:

git clone https://github.com/clarapereira/vdjveR.git

3. Prepare metadata table

4. Perform the alignment

5. Extract productive and DJ alignments from mixcr output

6. Merge all files in one table, add metadata

• export all, with metadata
• export productive clonotypes only
• export only productive TCR-B clonotypes

Rscript /path/to/vdjveR/mergeAlignmentFiles.R

7. Calculate Equitatibility

• plot data

Rscript /path/to/vdjveR/doClonotypicAnalysis.R
Rscript /path/to/vdjveR/doEquitabilityAnalysis.R