This article outlines the general workflow through the Biologics platform to analyze your variable region antibody and antibody-like sequences. It serves as a general guide and does not include all the options and edge-cases Biologics can accommodate. Please reach out to us if you are unsure if Biologics can process your data as we may be able to provide other solutions or work-arounds.
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Antibody Analysis
The above flowchart outlines the various ways you can analyze your sequences after pre-processing and annotation. The Biologics Annotator Result Document (shown above in pink) provides the stepping-off point for further analysis.
***Please see the pre-processing section of this article to learn more about how to process different sequences and whether Single Cell Analysis or Antibody Annotator will work best for your data.
The Biologics Annotator Result Document itself has a wealth of information for analysis, including:
- A Pipeline Report tab that can be useful for QA
- Clonotype/Cluster Tables
- More Graphs
- Specify filters to pull out sequences that meet certain metrics: Sequence filtering
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The Chain Combinations Table (Single Cell Antibody Analysis only)
- Biologics can group Heavy and Light chains that have the same barcode together and determine the proportions of different chain pairs within each barcode
- Biologics can group Heavy and Light chains that have the same barcode together and determine the proportions of different chain pairs within each barcode
What next?
After exploring your Biologics Annotator Result Document, there are multiple other ways you can further analyze and add to your data. Listed below are a selection of the most common:
- Adding Clusters after exploring your dataset
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Extract and Re-cluster to take a subset of sequences out of an existing Biologics Annotator Result Document and make a new document with re-calculated clusters
- It can be helpful to first filter for the sequences you want using our filtering tool
- Repair your sequences
- Compare two or more Annotation Result Documents from separate experiments to monitor clonal expansion etc.
- Add Assay Data to your sequences
- Perform an alignment on selected sequences to produce a tree
- Edit your sequences to make point mutations etc.
The videos in our Getting Started series may also be helpful, linked here. Below is our video on Understanding your Results in Sequence Data Tables.
Finally, you can export your annotated sequences as well as tables and graphs. See this article to learn about the different files you can export from Biologics.
Pre-Processing
Sanger Sequences and Processed Sequences
All these sequences will eventually feed into Antibody Annotator to produce an Annotation Result Document. You can learn more about Antibody Annotator and its various options here.
Links to the Pre-processing steps can be found below:
NGS and Single Cell/10X Datasets
NGS Sequences may go through NGS Antibody Analysis or Single Clone Antibody Analysis.
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NGS Antibody Analysis is designed to reduce your dataset by collapsing sequences together that are identical, or that meet a user-specified threshold of sequence similarity.
- NGS Antibody Annotator is also able to correctly handle and sort Barcoded data, or data with UMIs. See: Understanding Single Cell technologies: Barcodes and UMIs
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Single Cell Antibody Analysis is similar to NGS Antibody Analysis in that it collapses sequences, but it is optimised to take in Single Cell/10X data. It can both assemble reads and pair Heavy and Light chains within a cell/Barcode. See: Understanding Single Cell technologies: Barcodes and UMIs
- See NGS Tutorial 5 to learn how to process barcoded sequences that have already been partially demultiplexed (sequences are separated into sequence lists according to barcodes).
Links to the Pre-processing steps can be found below:
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Set Paired Reads
- Useful if not all read pairs are expected to overlap but you would like to retain this sequence information
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Merge Paired Reads
- Will pair and merge reads together using BBMerge
- Collapse UMI Duplicates and Separate Barcodes