Hello everyone! I wanted to ask if there is an example for Airrflow table for Bulk DNA BCR sequencing without the UMIs? Thanks

Hi @Aditee Kadam, what protocol did you use for amplification? Here is an example on how to run airrflow with a multiplex PCR amplification protocol without UMIs: https://nf-co.re/airrflow/4.3.0/docs/usage/#multiplex-specific-pcr-with-or-without-umi

I am using the Invivoscribe kit (no UMIs) with custom primers for BCR. I used the same command whose link you sent. I am running this Invivoscribe kit’s data with Airrflow. Since AIRRflow requires both V and C primers but we only have V and J primers, I used the J primer as the C primer input. The J primer position isn't absolutely fixed and varies by a few bases. Since most reads have the position as 33, I set it to 33. nextflow run nf-core/airrflow -r 4.2.0 -profile docker --input ./Metadata_test_airr.tsv --library_generation_method specific_pcr --cprimers C_primers.fasta --vprimers V_primers.fasta --cprimer_position R2 --outdir Airflow_results --umi_length 0 -c custom_resources.config --cprimer_start 33 --primer_revpr TRUE a) When I do set

--cprimer_start 33

, the pipeline fails at:

Error executing process > 'NFCORE_AIRRFLOW:AIRRFLOW:VDJ_ANNOTATION:CHANGEO_ASSIGNGENES (sample01)'
Caused by:
  Process `NFCORE_AIRRFLOW:AIRRFLOW:VDJ_ANNOTATION:CHANGEO_ASSIGNGENES (sample01)` terminated with an error exit status (1)

b) When I don't provide the

--cprimer_start

, the pipeline fails at:

PRESTO_MASKPRIMERS_SCORE_SANSUMI_REV
Missing output file(s) `*_primers-pass.fastq`

I think Airrflow cannot find the primers which leads to it having very few reads, and then the run fails. Could you please help me with this? Thanks.

I see, providing the J primer instead of C should work as well.Is the start position the position in the R2 reads or do you mean the position according to the germline? What airrflow expects is the start position of the primer in reference to the reads, so if the j primer starts at the beginning of the read you can provide 0, though this is the default and it also seems to fail. The issue might be what you mentioned that primers don’t always start at the same location. For the protocol without UMIs we don’t have right now the option to align the primers instead of expecting them at that particular position, as not so many people are using protocols without UMI, but we can implement it.

Thank you for your reply. I will send the fastq files and other details over mail.