Somatic hypermutation analysis

[pbpayal]

How to do somatic hypermutation analysis between two sample sets (3 samples per cohort) Can we do somatic hypermutation analysis from https SMARTer Mouse TCR a/b Profiling Kit? I came across "B cell lineage tracing" page.

How to create a diagram for mutation frequencies between two cohorts( no timepoints)?

[mizraelson]

Do you expect somatic hypermutations to be present in TCRs?

[pbpayal]

Extremely sorry. My mistake..I put the wrong kit. This is BCR sequencing data. (SMARTer Mouse BCR IgG H/K/L Profiling Kit)

[pbpayal]

I guess this is a very broad question. I am not an expert in immunology, so pardon my lack of in depth knowledge in this section. I see in the tutorial, a specific tree id is being chosen to do the tree reconstruction(https://mixcr.com/mixcr/guides/ig-trees-reconstruction/?h=trees#export-linage-trees-in-a-graphical-format). Whats the criteria to choose that specific tree id?

I have extracted the trees. What am I supposed to do after that? I want to look at the differences in WT vs Knockout mice?
`
mixcr findAlleles --report findAlleles.report.txt --export-alleles-mutations alleles.tsv --export-library alleles.json --output-template {file_dir_path}/{file_name}.group.reassigned.clns *.clns

mixcr findShmTrees --report Part1_trees.log *reassigned.clns Part1.shmt

mixcr exportShmTreesWithNodes part1_trees.shmt part1_trees.txt

`

[pbpayal]

And when I tried doing a random tree node, I get this error:

mixcr exportPlots shmTrees -m metadata_part1.tsv --node-color condition --ids 1424 part1_trees.shmt Mutant_shm_trees_1424.pdf

Error while running command shmTrees java.lang.RuntimeException: Duplicate library: repseqio.v4.0_with_found_alleles:10090

Attached metadata file.
metadata_part1.txt

[mizraelson]

Hi, sorry for the late reply.

To compare the SHMs status between two groups I suggest first run findAlleles and then:

mixcr exportClones \
-nMutationRate VRegionTrimmed \
-nMutationRate JRegionTrimmed \
input.clns output.tsv

This will return a repertoire table with the SHM rate (number of mutations divided by the gene segment length) for V and J genes. Then, you can take the average number across all clones in each sample and compare the two groups.

The trees make sense only within the same organism. I wouldn't recommend plotting a tree for multiple mice. The trees are rather used to track the B cell lineage over time (e.g., after vaccination or another event) in the same organism. In that case, the general approach is to pick a tree with a large number of nodes and look for one that has long branches.

[pbpayal]

Thank you for detailed explanation.