Generating Full TCR Alpha and Beta Sequences with stitchr

stitchr is a tool for reconstructing full-length T cell receptor (TCR) sequences from minimal input data. This guide focuses on using the command-line utility thimble, a wrapper included with stitchr, for batch processing of TCR alpha and beta chains.

The Everocode^TM TCR assay was optimized to capture the CDR3 information from TRA and TRB chains and confidently determine the paired clonotype information. The assay achieves sufficient coverage across the VDJ gene segments to provide VDJ annotations along with the CDR3 information for the TRA and TRB chains- that will include the gene-segment annotations for TRBV : VDJ , TRAV:VJ and also the TRAC and TRBC. The Parse bioinformatics pipeline generates TCR output files such as tcr_annotation_airr.tsv, in standardized AIRR format, which contains the identified contigs in each cell along with their VDJ chain annotations. The tcr_annotation_airr.tsv file can be utilized by the stitchr tool to predict and stitch together germline annotations with the CDR3 sequences to output the full-length coding nucleotide sequences for TRA and TRB chains.

Prerequisites

Before you begin, ensure you have installed stitchr and the necessary data (for the genome of interest) by following the instructions here.

Input File Format

We are going to use thimble wrapper provided by stitchR to run the analysis. thimble expects the input file to have a specific format (see below).

TCR_name TRAV TRAJ TRA_CDR3 TRBV TRBJ TRB_CDR3 TRAC TRBC TRA_leader TRB_leader Linker Link_order TRA_5_prime_seq TRA_3_prime_seq TRB_5_prime_seq TRB_3_prime_seq

We will be utilizing the tcr_annotation_airr.tsv output file from the Parse split-pipe TCR pipeline (using --tcr_analysis parameter) as a starting point. We will analyze TRA and TRB chains separately. The following command will format the output from tcr_annotation_airr.tsv into the expected format for TRA and TRB chains.

#For extracting TRA chains into the expected format
awk -F'\t' 'NR==1 {
    print "TCR_name\tTRAV\tTRAJ\tTRA_CDR3\tTRBV\tTRBJ\tTRB_CDR3\tTRAC\tTRBC\tTRA_leader\tTRB_leader\tLinker\tLink_order\tTRA_5_prime_seq\tTRA_3_prime_seq\tTRB_5_prime_seq\tTRB_3_prime_seq"
}
NR>1 && $3=="TRA" {split($8, J, "_");val=$6;split(val, V, "_");val = V[1];
gsub(/-?DV/, "/DV", val);gsub(/-?OR/, "/OR", val);$6=val;
    print $1 "\t" $6 "\t" J[1] "\t" $14 "\t \t \t \t" $9
}' /PATH/TO/tcr_annotation_airr.tsv > thimble_TRA_input.tsv
#For extracting TRB chains into the expected format
awk -F'\t' 'NR==1 {
    print "TCR_name\tTRAV\tTRAJ\tTRA_CDR3\tTRBV\tTRBJ\tTRB_CDR3\tTRAC\tTRBC\tTRA_leader\tTRB_leader\tLinker\tLink_order\tTRA_5_prime_seq\tTRA_3_prime_seq\tTRB_5_prime_seq\tTRB_3_prime_seq"
}
NR>1 && $3=="TRB" {split($8, J, "_");val=$6;split(val, V, "_");val = V[1];
gsub(/-?DV/, "/DV", val);gsub(/-?OR/, "/OR", val);$6=val;
    print $1 "\t \t \t \t" $6 "\t" J[1] "\t" $14 "\t \t" $9
}' /PATH/TO/tcr_annotation_airr.tsv> thimble_TRB_input.tsv

Running thimble

Once you have the input file, you can run thimble using the command below.

#For analyzing TRA sequences
thimble -i thimble_TRA_input.tsv -o thimble_TRA_output.tsv -r a -s HUMAN
#For analyzing TRB sequences
thimble -i thimble_TRB_input.tsv -o thimble_TRB_output.tsv -r b -s HUMAN

Required Parameters

• -i: Input file path
• -o: Output file path
• -r: Kind of TCR, i.e. a/b or g/d
• -s: Species, i.e HUMAN or MOUSE

Additional options can be found here.

Output File Format

The output is a TSV file with these key columns: