Detection T cell receptor transcripts

Question

[jsw940]

Hello, I planned to explore diverse T cell receptor transcripts using long-read sequencing. So I transfected TCR mini gene into cells and got sequencing data.

I mapped my data to genome reference and visualize it via IGV. But I couldn't see any transcripts from TCR plasmid on the chromosome 7 where T cell receptor locus is.

How can I detect reads from transfected plasmid? Shall I make custom reference file using plasmid map?

Or should I use any specific ways to detect TCR transcripts like IGMT, MiXCR, IgBLAST and so on?

I just want to see the diversity of splicing patterns using tools like FLAIR.

I need your advice! Thank you.

[Andrew]

Did you find any solution? I'm also trying to find T cell receptor expression in NGS transcriptome data, and it doesn't seem like it's represented anywhere. It's gotta be there somewhere, right?

[jsw940]

Hi Andrew. I just used my plasmid map as a custom reference. So I can not advise you :( But I got some information from papers using ImmunoSeq. I think maybe there is some information for you!

Answer 1

[caggtaagtat]

Hi, as you said, you could add the sequence of your minigene to your reference fasta. After uploading it to IGV, there will be a new option in the chromosome select field to view reads mapping on the mini-gene.

Maybe you have artificial subsequences in your minigene, which makes it different from the original locus.

Answer 2

[mizraelson]

Since the TCR locus is rearranged (compared to the genome in other cells) in mature receptor genes you should use a specialized software to reconstruct the CDR3 region and rearrangement.

You can use MiXCR for that. We have recently added support for long-read data also.

If you can give more details on the sequencing platform and library structure I can help you with running MiXCR.

Feel free to ask us on github: https://github.com/milaboratory/mixcr

or e-mail:

[e-mail]

In general it shouldn't be hard, e.g.:

mixcr analyze generic-tcr-amplicon \
--species mmu \
--rna \
--rigid-left-alignment-boundary \
--floating-right-alignment-boundary C \
  input_R1.fastq.gz \
  input_R2.fastq.gz \
  result

But certain library structures (or long reads sequencing platform) might need some tweaks. You can also check our new documentation portal: https://docs.milaboratories.com/