TCR data analysis

Question

[bthomas4]

We are using Takara a/b TCR profiling kit. The libraries were run on MiSeq 600 and we got 600,000 to 1400,000 reads per sample. Before analysis using MIXCR, should the reads be downsampled? Should negative and positive controls be included in the analysis? What are the best options for trimming and analysis? We are looking for TCR diversity and clonality.

[GenoMax]

Page 23 of Human TCR a/b profiling kit manual has some advice.

Answer 1

[mizraelson]

Hi, First of all, please check our new MiXCR 4 which has a dedicated support for Takara kits (including UMI support for kits with molecular barcodes).

e.g.

mixcr analyze takara-human-bcr-cdr3 \
  input_R1.fastq.gz \
  input_R2.fastq.gz \
  result 

More on that here: https://docs.milaboratories.com/mixcr/reference/overview-built-in-presets/#takara-bio

To answer your questions: 1) No, you dont need any additional preprocessing before MiXCR. Sometimes you can use some normalization after you get clonotype tables: to the number of reads or UMI for example. More on that here: https://docs.milaboratories.com/mixcr/reference/mixcr-downsample/

2) I would include both positive and negative controls to ensure that you negative control does not have any clones and positive control returns the expected result.

3) You can use MiXCR postanalysis directly to access clonality and diversity metrics. More on that here: https://docs.milaboratories.com/mixcr/reference/mixcr-postanalysis/#diversity-measures