Scripts for our functional TCR screening paper: Fahad, A.S., Chung, C.Y., López Acevedo, S.N. et al. Cell activation-based screening of natively paired human T cell receptor repertoires. Sci Rep 13, 8011 (2023). https://doi.org/10.1038/s41598-023-31858-4

Requirements

• FASTX-Toolkit v0.0.14
• MiXCR v2.1.12
• USEARCH v5.2.32
• A UNIX-like system

Data

Raw TCRα:β MiSeq data have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA827461.

Usage

1. Perform quality control with qualityfilter_script_human_v1.1.sh for forward and reverse reads. This script requires FASTX-Toolkit v0.0.14. A quality threshold of q20p50 was used. A prefix name should be selected to match file names in the next steps.

   bash qualityfilter_script_human_v1.1.sh forward_reads.fastq reverse_reads.fastq prefix

2. Anotate reads using jobsubmit-2.sh for TRAV and jobsubmit-3.sh for TRBV genes. These scripts require MiXCR v2.1.12.

   bash jobsubmit-2.sh reverse_reads.fastq-2 prefix
   bash jobsubmit-3.sh forward_reads.fastq-1 prefix
   

3. Once gene annotation is complete, we can stitch paired-end reads using stitch_TRA-TRB.sh

   bash stitch_TRA-TRB.sh prefix

4. Generate pair results by combing the reads of alpha and beta into complete chain information in one file based on mixcr sequence IDs. This is done by processing.sh script.

   processing.sh prefix

5. Reads are prepared for clustering using CDR3motif_search_v1.sh. This will aggregate reads with the same CDR3B nt/aa sequences and reports the number of reads. Next, it filters out clones with only one read.

   CDR3motif_search_v1.sh prefix

6. Next, reads are clustered based on CDR3 identity using CDR3motif_search_analysis_v3.1.sh. This step requires USEARCH

   bash CDR3motif_search_analysis_v3.1.sh prefix raw_reads

Example

1. Quality filter:

   bash qualityfilter_script_human_v1.1.sh Exp-name_R1_001.fastq Exp-name_R2_001.fastq Exp-name

2. Run MIXCR

   bash jobsubmit-3.sh Exp-name_r1_q20p50_filtered.fastq-1 Exp-name
   bash jobsubmit-2.sh Exp-name_r2_q20p50_filtered.fastq-2 Exp-name
   

3. Stitch paired-end reads

   bash stitch_TRA-TRB.sh Exp-name

4. Reformate files

   bash processing.sh Exp-name

5. Prep for CDR3B clustering

   bash CDR3motif_search_v1.sh Exp-name

6. Run clustering script

   bash CDR3motif_search_analysis_v3.1.sh Exp-name Exp-name_nt_pairs_over1read.txt

7. Exp-name_unique_final.txt contains the final estimated Alpha:beta clonotypes

   ls | grep unique_final | xargs wc -l