This article describes how to use the Pair Heavy & Light Chains pipeline. The below video gives a general introduction to pre-processing in Biologics. The first few videos in our Getting Started series may also be helpful, linked here.
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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.
There are two ways to pair chains in Biologics:
- Manually using Pre-Processing > Pair Heavy/Light Chains
- Described below in this article
- Automated using Pre-Processing > Batch Assemble Sanger Sequences
- This allows you to assemble forward and reverse reads automatically and to pull out the information in your sequence names to associate Heavy and Light chains together
- Please see our other article Using Name Schemes to Pair Chains and Assemble Sequences to follow this method.
Running Pair Heavy/Light Chains
- Navigate to your folder with input data (or upload the data),
- Select all of the files in the folder, or Group the Sequences into a sequence list first (recommended if you have more than 50 sequences)
- Click Pre-processing and
- Select Pair Heavy/Light Chains in the dropdown.
Options
- 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. See How to Create a Name Scheme for more information. The video below explains this concept:
- 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.
Sequence Annotation
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. See our article Antibody Annotator for more information.