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| # MiXCR Shotgun Analysis
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| Collection of tools to run MiXCR on shotgun (RNA-Seq and WES) samples and analyze them. Analysis tools for clonality, heavy/light chain ratio (pro-B signature) and VDJ gene usage heatmaps. | | Collection of tools to run MiXCR on shotgun (RNA-Seq and WES) samples and analyze them. Analysis tools for clonality, heavy/light chain ratio (pro-B signature) and VDJ gene usage heatmaps. |
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| Git repository: https://gitlab.com/guilhermenngiusti/mixcr_shotgun | | Git repository: https://gitlab.com/guilhermenngiusti/mixcr_shotgun |
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| ## How to use
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| ### Running MiXCR
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| MiXCR docker container can be obtained using the following command:
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| `docker pull milaboratory/mixcr`
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| MiXCR analysis of RNA-Seq or WES can bu run using the following command:
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| `docker run -it --rm -v $(pwd)/VOLUME_DIR:/work milaboratory/mixcr:latest mixcr analyze shotgun -s mmu --starting-material MATERIAL R1_FASTQ R2_FASTQ WORK_DIR`
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| ### Clonality analysis
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| The Python script to perform clonality analysis can be run from the top level directory using the following command:
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| `python3 tools/clonality.py -i SAMPLE_DIR -o OUTPUT_DIR -m TARGET_MARKER`
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| Argument description:
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| - `-i` Directory where MiXCR clonotypes files are stored;
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| - `-o` Directory for output files;
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| - `-m` Target marker. Options: `IGH`, `IGK`, `IGL`, `TRA`, `TRB`, `TRD` and `TRG`;
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| - `-t` (Optional) Translation table used to rename samples. Table must be a `tsv` file with the columns `original_name` and `translated_name`.
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| Each wedge of the resulting plot will only be labeled if its clonotype presents frequency ≥ 5%.
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| Gini-Simpson diversity index presented at the center of the plot.
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| This script also produces a `count.txt` file containing the total number of reads for the analyzed marker.
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| ### Pro-B cell signature analysis
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| The Python script to perform pro-B cell signature analysis can be run from the top level directory using the following command:
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| `python3 tools/pro-B_signature.py -i SAMPLE_DIR -O OUTPUT_DIR -n ANALYSIS_TITLE`
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| Argument description:
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| - `-i` Directory where MiXCR clonotypes files are stored. Each sample contained in this directory must be stored in its own subdirectory;
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| - `-o` Directory for output files;
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| - `-n` Title for the analysis. Used as the name for the `jpg` file generated;
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| - `-t` (Optional) Translation table used to rename samples. Table must be a `tsv` file with the columns `original_name` and `translated_name`;
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| - `--yscale` (Optional) Max value for the ordinates axis. Default `10`.
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| ### VDJ genes usage analysis
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| The Python script to perform VDJ genes usage analysis can be run from the top level directory using the following command:
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| `python3 tools/vdj_heatmap.py -i SAMPLE_DIRS -o OUTPUT_DIR -m MARKER -g GENE_FAMILY`
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| Argument description:
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| - `-i` Directory where MiXCR clonotypes files are stored. Can receive multiple directories;
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| - `-o` Directory for output files;
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| - `-m` Target marker. Options: `IGH` or `IGK`;
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| - `-g` VDJ gene family. Options: `V`, `D` and `J`;
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| - `--xsize` (Optional) Heatmap width;
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| - `--ysize` (Optional) Heatmap length;
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| - `-t` (Optional) Translation table used to rename samples. Table must be a `tsv` file with the columns `original_name` and `translated_name`;
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| - `-l` (Optional) Draws horizontal lines separating sample groups in heatmap. Integer representing after how many sample the line should be drawn. Can receive multiple values;
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| - `--linewidth` (Optional) Sets width for horizonal lines separating sample groups in heatmap;
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| - `-s` (Optional) Sample to be represented on a secondary heatmap. Can receive multiple values. If a translation table is used, the provided sample name should be the translated one;
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| - `-S` (Optional) Group name for separate samples, used for secondary heatmap output file;
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| - `--secxsize` (Optional) Secondary heatmap width;
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| - `--secysize` (Optional) Secondary heatmap length.
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| The heatmaps generated by this script always represent only one marker (e.g. IGH) and VDJ gene family (e.g. V) at a time.
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| **This script currently only supports the IGH and IGK markers from mice.** In order to perform analysis on other markers, complete genome coordinate files should be added to `tools/coordinates`, using the same format as the `tsv` files already available. In order to analyze human (or other species) markers, replace the genome coordinate mice files for ones from your species.
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| Genomic coordinates files can be generated at https://www.ncbi.nlm.nih.gov/datasets/gene/.
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| ## Credits
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| Created by Guilherme Giusti at Boldrini Research Center, 2023.
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