AmpliCDR3 - Amplicon Sequencing T-cell CDR3 repertoire analysis tool
AmpliCDR3 extracts and analyses T-cell receptor (TCR) CDR3 region sequences.
AmpliCDR3 processes reads from NGS targeted/amplicon sequencing of TCR repertoires to extract and analyze their CDR3 regions.
CDR3 region boundaries are indicated by the presence of a highly conserved TG[TC][GA][CG] motif that marks the end of V segment and the beginning of the CDR3, and a fixed distance from the end of the CDR3 to the 3' primer/sequence located in the Constant region (mandatory parameter 'primer_r_dist' within the 'amplicon data' CSV file).
AmpliCDR3 is able to analyze data from any non-model vertebrate species, without any reference sequences for the V and J segments, as long as we can define a protein pattern to recognize the boundaries of the CDR3 region pattern and indicating the expected distance from the 3’ end of the CDR3 and the C region primer sequence.
Inherent sequencing error rates of the NGS techniques are crucial during the repertoire analysis because they may artificially inflate the CDR3 diversity in the sample. To solve this caveat, AmpliCDR3 performs filtering (e.g. removal of singletons) and clustering of CDR3 sequences based on their similarity. It can help remove artefacts, such as sequencing errors, PCR errors or PCR chimeras - but this option must be used with caution, as clusters may include several highly similar, true CDR3 variants.
Currently, the most efficient way to correct NGS (and PCR) errors in immune repertoire profiling is to implement molecular barcoding with Unique Molecular Identifiers (Shugay et al. 2014). AmpliCDR3 can process reads with UMIs (presence of which have to be specified in the 'amplicon data' CSV file as multiple 'N's between parentheses). CDR3 variants with the same UMI are grouped together, and a consensus sequence is retrieved per each UMI cluster (when possible).
AmpliCDR3 requires as input:
- SEQUENCE FILE *: Reads from the experiment in a FASTQ or FASTA format file (compressed or uncompressed).
- AMPLICON DATA: Primer and tag information in a CSV format file as explained in the documentation.
Results can be downloaded on the same page or from an email message after analysis completion.
For more information, read the documentation.