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Hi everyone, I am new to analyzing AIRR-seq data a...
# airrflow
f
Hi everyone, I am new to analyzing AIRR-seq data and to nf-core/airrflow, so I hope this is the right place to ask for help. I am trying to analyze data generated with the Takara Bio MaxiSeq Human FFPE TCR+BCR (with UMIs) kit (https://www.takarabio.com/products/next-generation-sequencing/immune-profiling/human-repertoire/maxiseq-human-ffpe-tcrbcr-(with-umis)) from FFPE tumor samples So my questions are: 1. Does anyone have experience running airrflow with this specific Takara Bio MaxiSeq FFPE kit, and could you share what primer FASTA files / parameters you used? 2. Regarding the primer FASTA files themselves: the kit does not provide the C-region primer sequences explicitly in the user manual, and there are no V-region primers in the PCR design (the kit uses a universal extension primer instead). How would you recommend obtaining or constructing the 
--cprimers
and 
--vprimers
files needed for the analysis? Is there a public resource or a standard workaround for commercially closed primer pools like this one? Any guidance would be greatly appreciated. Thanks in advance!
h
Hi @Francisco Martínez Picó, we have no previous experience with this protocol. I need some time to learn it before giving you a more detailed answer. I will reach out to you afterwards. BTW, did the fastq files you get for your samples have BCRs and TCRs combined or separated? nf-core/airrflow is not able to process BCRs and TCRs together in one run.
f
Thanks so much for your answer. As far as I know, this kit is pretty new. I would expect the FASTQ files to contain both TCR and BCR reads, so maybe airrflow is not the best choice. I will try the manufacturer's software to analyze them.
Thanks again. 🙂
g
Hi @Francisco Martínez Picó, to extend a bit more on your answer. We included some other TAKARA Bio protocols as presets to the pipelines. One common way to go about protocols that do not provide the C region primers, is to detect universal C region primers, this needs to be validated on a protocol-by-protocol basis, but of the TAKARA SMART-seq and SMARTer-seq protocols this works well for us. The problem with this protocol is that it combines TCR and BCR sequences into one sample, and it is not possible to analyze BCR and TCR sequences in the same airrflow run due to the clonal analysis step. The pipeline currently allows only to provide one set of settings for the clonal per run, and TCRs and BCRs need different settings (clonal_threshold 0 for TCRs and >0 for BCRs).
Here is an example on how to analyze samples from the TAKARA SMART-seq kit: https://nf-co.re/airrflow/5.0.0/docs/usage/#takara-smart-seq-human-bcrtcr-profiling-kit
And here is a list of the params that this profile sets: https://github.com/nf-core/airrflow/blob/master/conf/takara_smartseq_umi_bcr.config
You can however run the pipeline twice on the same samples, once for processing the TCR sequences (provide 
pcr_target_locus
TR in the samplesheet), and once for the BCR sequences (provide 
pcr_target_locus
IG in the samplesheet). You can test whether the universal C primers that we provided for the other TAKARA protocols work for this one as well. The command would then be:
Copy code
nextflow run nf-core/airrflow -r 5.0.0 \
-profile docker \
--input input_samplesheet.tsv \
--outdir results -c custom.config
Where you have the following contents in the custom config:
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/*
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
    Nextflow config file for running minimal tests
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
    Defines input files and everything required to run a fast and simple pipeline test.

    Use as follows:
        nextflow run nf-core/airrflow -profile takara_smartseq_umi_bcr,<docker/singularity> --outdir <OUTDIR>

----------------------------------------------------------------------------------------
*/

params {
    config_profile_name        = 'Takara SMART-Seq umi BCR'
    config_profile_description = 'Profile to run pipeline for the Takara SMART-Seq (UMI) BCR protocol profile'

    mode = 'fastq'

    library_generation_method = 'dt_5p_race_umi'

    cprimers = '<https://github.com/immcantation/immcantation/raw/refs/heads/master/protocols/Universal/Human_IG_CRegion_RC.fasta>'

    // primer options
    cprimer_position = 'R1'
    cprimer_start = 0
    vprimer_start = 0
    umi_length = 12
    umi_position = 'R2'
    cluster_sets = false


    // Mask primer options
    maskprimers_align_race = true
    primer_r2_extract_len = 7
    primer_r1_mask_mode = 'cut'
    primer_r2_mask_mode = 'cut'
    primer_maxlen = 70
    primer_r1_maxerror = 0.2
    assemblepairs_sequential = true
    primer_consensus = 0.6
}
And here you can edit if needed the parameters according to your protocol. The important things to check would be, where is the UMI located (R1 or R2) and what is the length of the UMI barcode for this protocol, what is the length and position of the RACE linker (here it is in the R2 read, and the length is specified in the 
primer_r2_extract_len
param, and confirm that the Cprimer position is also in R1 for your protocol
f
Thanks so much for your help 🙂
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