Analyzing TCR sequences in Geneious Biologics

Jump to:

• How TCR sequences are represented in Geneious Biologics
• Auto-annotating your TCR germline genes to make a Database
  • Curating the output
    
• Manually annotating TCR germline genes to make a Database
  • Overview
  • Correctly formatting the Germline Genes
    
    

How TCR sequences are represented in Geneious Biologics

By default, Biologics determines the chain by checking the first three letters of the gene name. Antibody Annotator and Single Clone Analysis will label chains accordingly:

- TRB and TRD chains as Heavy chains
- TRA and TRG chains as Light chains

Note that Biologics will only pair TRA and TRB chains together, or TRG and TRD chains together. We do allow recombination between certain V and J genes from the TRA and TRD families, as the V and J recombination mechanisms are less strict in TCRs.

Most of the help articles written with Ig sequences in mind will still apply for TCR sequences, as their variable regions have a similar layout.

Auto-annotating your TCR germline genes to make a Database

NOTE: The output format of your germline sequences using this method is at best an approximation. The correct annotation types will be added automatically to your sequences, however the boundaries of the CDR/FRs may be incorrect. We strongly advise you inspect and curate the output.

First, you will need to obtain the reference sequences. Before uploading to Geneious Biologics, your sequences will need to be arranged into Separate sequence lists:

Heavy Sequences
TRB and TRD gene sequences should be broken up into at least three separate sequence lists:

• A sequence list containing all the Heavy V Genes. This will contain all the V genes from the TRB and TRD gene families.
  • Name this sequence list "IGHV"
• A sequence list containing all the Heavy D Genes. This will contain all the D genes from the TRB and TRD gene families.
  • Name this sequence list "IGHD"
• A sequence list containing all the Heavy J Genes. This will contain all the V genes from the TRB and TRD gene families.
  • Name this sequence list "IGHJ"
• Optional: A sequence list containing all the Heavy C Genes. This will contain all the C genes from the TRB and TRD gene families. 
  • Name this sequence list "IGHC"

Light Sequences
TRA and TRG gene sequences should be broken up into at least two separate sequence lists:

• A sequence list containing all the Light V Genes. This will contain all the V genes from the TRA and TRG gene families.
  • Name this sequence list "IGLV" or "IGKV"
• A sequence list containing all the Light J Genes. This will contain all the J genes from the TRA and TRG gene families.
  • Name this sequence list "IGLJ" or "IGKJ"
• Optional: A sequence list containing all the Light C Genes. This will contain all the C genes from the TRA and TRG gene families.
  • Name this sequence list "IGLC" or "IGKJ"

To auto-annotate your list(s) of genes, navigate to the Reference Databases on the left panel and click on the 3 vertical dots to bring up the New database option:

Next, follow the prompts to upload your reference sequences. Make sure you have selected Annotated Germline as the Database type and that the option to Automatically format germline genes is selected.

In the next step, select an existing gene reference database as the basis for estimating the FR/CDR boundaries etc. Choosing a reference database from a species more closely related to your target sequences will have better results - however, the output will still require curation.

Make sure to select Sequence List Names as the Gene name source:

After clicking next, a summary of the Reference Database to be created will be displayed. The option to proceed will not be available until you have confirmed that you understand the need for inspecting and curating the output by selecting the following box: I understand that I need to inspect and curate the formatted output. 

Curating the output

Biologics will attempt to place various annotations on the raw germline sequences, according to rough estimates of the CDR and FR boundaries as determined by the chosen reference database. However, this method will not necessarily produce scientifically correct region boundaries. Expert knowledge of the sequences will need to be applied to curate the output.

To curate and inspect the output germline reference database, Export the output to a sequence file. Make sure you select a file type that will preserve the annotations, like .geneious. You can then inspect and edit the annotations in Geneious Prime.

In particular, a few assumptions are made by Biologics when generating the formatted output:

• Constant genes will be assumed to start in reading frame 3. If your constant regions are in frame (ie. start at the beginning of the sequence), you can adjust your results by deleting the qualifier codon_start: 3 from the C_segment annotation on constant gene sequences. This is shown below in Geneious Prime when editing the annotations:
  
  You can also edit this qualifier to codon start: 2 if the true reading frame starts from the second nucleotide.
  
  
• The CDR/FR boundaries and the general length of all annotations are estimated based on the existing reference database chosen. This means they may need adjusting. We recommend inspecting each individual sequence to check if the annotations begin and end where you expect.

For more information on what format the annotations need to be in, you can refer to the section below.

Manually annotating TCR gene sequences to make a Database