Custom milab-human-rna-tcr-umi-race about mixcr HOT 5 CLOSED

Yes, what barcode sequence do you have?

from mixcr.

We have followed 5'RACE protocol, however, we have in R1 ^NNNNNCAGTGGTATCAACGCAGAG-UNNNNUNNNNUNNNNU-CTT, and in R2 ^NNNNNACACNNNNTTCAGGTCCTC. Really happy for help! Thanks!

from mixcr.

As I understand your UMI is in R1. What is the NNNN sequence in the R2?

from mixcr.

Yes, it is right! NNNNN - random nucleotides which introduced at 5’-ends of the final library for better diversity generation and cluster differentiation by Illumina sequencer. And the second NNNN in read one is STTK (listed as that sequence, but pretty random among the files).

from mixcr.

I recommend trying the following command:

mixcr analyze generic-amplicon-with-umi \
    --species hsa \
    --rna \
    --tag-pattern "^N{5}cagtggtatcaacgcagag(UMI:N{16})ctt(R1:*)\^N{5}acacN{4}ttcaggtcctc(R2:*)" \
    --rigid-left-alignment-boundary \
    --floating-right-alignment-boundary C \
    --assemble-clonotypes-by VDJRegion \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

The --assemble-clonotypes-by VDJRegion parameter suggests that your reads cover the full receptor sequence. Omit this parameter if you are using short reads, for example, 150bp+150bp.

from mixcr.