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, you can choose to pair sequences using one of four methods:
Interlaced sequences within each list: Sequences will be paired by order in the list(s) selected. The first two sequences in each list will be paired with one another, then the next two and so on.
Pairs of lists: The first sequence in the first list will be paired with the first sequence in the second list and so on for even numbers of lists.
Name scheme: The name scheme selected will be applied to the sequence names to extract the Common Identifier field which will be used for pairing. When two sequences have the same Common Identifier, they will be paired together. For more information about name schemes, see this article.
Match sequence names (for standalone sequences, or within each list) (slow): This method attempts to pair similar sequence names based on single character differences between the names. It expects that sequence names 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, as the heavy and light chain sequences name only differ by a character (VH and VL). However 123_VH_myPrimer.ab1 and 123_VL_myOtherPrimer.ab1 would not pair together as they differ by more than one character. If needed, users can run the Batch Rename preprocessing operation first to rename sequences in a consistent manner before running Pair Heavy/Light Chains.
Note: Using the “Name Scheme” method above for pairing Heavy/Light Chains is usually preferable, as it is more flexible and produces more predictable results.
Click Run to start the operation. This operation will produce a Nucleotide sequence list document.
To check whether the sequence chains are paired, in your folder select the paired Sequences, and 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 "Selected Sequences are: 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.