This article describes how you can take individual heavy and light chain sequences, and pair them for downstream sequence annotation and analysis. The article covers chain pairing, running Antibody Annotator analysis, adding external assay data and sequence alignment.
Heavy and light chain pairing
Pairing heavy and light chain sequences from a clone prior to downstream analysis enables you to analyze both chains as one clone. To pair the heavy and light sequences,
- Navigate to your folder with input data (or upload the data),
- Select all of the files in the folder,
- Click Pre-processing and
- Select Pair Heavy/Light Chains in the dropdown.
In the Pair Heavy/Light Chains dialog, select Match sequence names (for standalone sequences, or within each list) (slow).
Note: "Match sequence names" expects that sequences are mostly consistent and the heavy and light chain sequences have the same name except for a single variation, for example 123_VH.ab1 and 123_VL.ab1 would pair together, but 123_VH_myPrimer.ab1 and 123_VL_myOtherPrimer.ab1 would not. If needed, users can run the Batch Rename preprocessing operation first to rename sequences in a consistent manner before running Pair Heavy/Light Chains.
Click Run to start the operation. This operation will produce a Nucleotide sequence list document.
As the heavy and light chain sequences name only differ by a character (VH and VL) they will be paired together. To check whether the sequences are paired by name, navigate to 2. Heavy-Light chain pairing folder, (2) select Sequences (paired) and (3) hover over the sequence name or the sequence.
The red boxes indicate paired sequences and there should be a total of 137 heavy and light chain pairs matched by sequence name. In the example above, abituzumad_VH and abituzumad_VL are a pair.
To annotate and analyze the paired sequences, select Sequence (paired) document, click Annotation and select Antibody Annotator in the dropdown.
You will want to select the option "Both Chains in Associated Sequences" in order to ensure that your two chains are recognised as individual chains and analysed together. For more information on Antibody Annotator options, look here.