Workflow: NGS Antibody Analysis

If you regularly run Set & Merge Paired Reads before running NGS Antibody Annotator, the NGS Antibody Analysis workflow lets you configure and run both steps as a single operation. Rather than waiting for each step to complete before setting up the next, you configure all your options upfront and receive annotated and clustered result files when the entire analysis is finished.

You can also use the workflow to batch-analyze multiple datasets in a single run, producing separate Annotation Results for each dataset.
 

Jump to:

• What is NGS Antibody Analysis?
• How do I run NGS Antibody Analysis?
• Settings
  • Step 1: Set & Merge Paired Reads
  • Step 2: Annotation
  • Step 3: Output
• Single result vs. separate results (batching)
• Applying saved settings as Profiles
• Viewing your results
   

What is NGS Antibody Analysis?

NGS Antibody Analysis is a workflow pipeline that enables the Set & Merge Paired Reads and NGS Antibody Annotator pipelines to be run as a single operation. You can configure options for each step in the wizard, click Run, and receive a Biologics Annotation Result document without needing to monitor and trigger each step manually.

This is useful when your input data consists of R1 and R2 read pairs that need to be set and/or merged before annotation. The workflow handles the intermediary steps automatically - reads can be either set or merged before being passed directly to NGS Antibody Annotator.

The workflow also supports batching: if you have multiple datasets (e.g. four samples, each with an R1 and R2 file), you can choose to produce either a single combined result or separate Annotation Results - one per dataset - all from a single run.

The workflow produces the same Biologics Annotation Result(s) as running the NGS Antibody Annotator standalone. All the same analysis capabilities are available, including germline matching, liability detection, clonotypes, clustering, and the full visualization suite. For details on what the annotator does, see NGS Antibody Annotator.
 

How do I run NGS Antibody Analysis?

Select your input file(s) within a folder and go to Run Workflow > NGS Antibody Analysis:

This opens a step-through wizard where you configure options for each step in the workflow. The wizard has three steps: Set & Merge Paired Reads, Annotation, and Output. Navigate between steps using the Next and Back buttons.

When you are satisfied with your settings, click Run on the final step to start the workflow.
 

Settings

The wizard presents each step of the workflow with its own set of options. These are the same options available in the corresponding standalone pipelines, so if you are already familiar with those, the settings will be the same here.

Each step allows you to select a previously saved Profile for that pipeline. See Using Profiles below.
 

Step 1: Set & Merge Paired Reads

This step configures how your reads are paired and merged. If your reads are already paired or merged, select Skip this step and the workflow will pass your input files directly to the Annotation step.

If you have a saved Profile for Set & Merge Paired Reads, you can choose this here. See Using Profiles below.

Pair by:

• Pairs of lists 
  Suitable for R1 and R2 read lists. This option is only available when an even number of sequence lists is selected.
• Interlaced sequences in each list 
  Pairs alternating sequences within each list.

Read Orientation:

Specifies the orientation of your paired reads. Options include Forward/Reverse (Illumina paired ends), Reverse/Forward (Illumina mate pairs), and Forward/Forward (SOLiD).

Merging:

Controls how paired reads are merged.

• Set paired reads only 
  Pairs reads without merging them. When selected, the Expected length of sequenced region option is enabled.
  • Choose this setting if you are experiencing low merge rates, as the NGS Antibody Annotator can merge reads more accurately using the built in logic for V(D)J regions.
• BBMerge Default settings 
  Merges overlapping paired reads using BBMerge with default sensitivity.
• BBMerge Slow 
  Decreases false positives during merging.
• BBMerge Fast 
  Increases false positives during merging.
• BBMerge Very Fast 
  Greatly increases false positives during merging.

When any BBMerge option is selected, the Iteratively trim option becomes available.

For full details on all Set & Merge Paired Reads options, see Set & Merge Paired Reads.
 

Step 2: Annotation

This step configures the NGS Antibody Annotator. The options are identical to those available in the standalone NGS Antibody Annotator pipeline. The Main Options section is expanded by default, while Analysis Options, Filtering Options, Clustering Options, and Advanced Options are collapsed.

As with the previous step, you can select a saved Profile for the annotator. See Using Profiles below.

For full details on all NGS Antibody Annotator options, see NGS Antibody Annotator.
 

Step 3: Output

Output folder name: 
Optionally specify a name for the output folder where results will be saved. If left blank, results are saved in the same folder as your input files.

Analyze as:

This option determines whether your input datasets are combined or analyzed independently.

• Single Annotation Result 
  All selected input datasets are combined and analyzed together, producing one Biologics Annotation Result. This is the default option.
• Separate Annotation Results (one for each dataset) 
  Each dataset is analyzed independently, producing a separate Biologics Annotation Result per dataset. See Single result vs. separate results (batching) below for details on how datasets are defined.

Overview of Results: 
A summary of the expected output, including how many Annotation Results will be produced from your input documents and any additional intermediary documents (e.g. lists of unmerged reads). Review this to verify that the workflow is configured as expected before running it.

Click Run to start the workflow.
 

Single result vs. separate results (batching)

When you select Single Annotation Result, all input files are merged and annotated together into one Biologics Annotation Result, regardless of how many sequence lists you selected. The same settings are applied to everything.

• Note: sequences are only collapsed within lists/datasets, not across lists/datasets

However, when you select Separate Annotation Results, the workflow treats each dataset independently:

For NGS Antibody Analysis, a dataset is defined as follows:

• If using interlaced read pairs or full V(D)J sequences in each sequence list, each sequence list is treated as a separate dataset.
• If using R1 and R2 Illumina read files, each R1 and R2 pair for the same sample represents one dataset.
  • The R1 and R2 reads within a dataset are paired and merged only with each other - the R1 read from one sample will not be merged with the R2 read from a different sample.

Each dataset/sequence list is then passed independently to the NGS Antibody Annotator.

All datasets in a batched run use the same settings for every step in the workflow.

The Overview of Results in the Output step will show the expected number of Annotation Results, so you can confirm the workflow has correctly identified your datasets before clicking Run.
 

Applying saved settings as Profiles

Each step in the wizard supports Geneious Biologics Profiles. If you have previously saved profiles for Set & Merge Paired Reads or NGS Antibody Annotator, you can select them at the top of the relevant step using the Profile dropdown. 

This is useful for lead scientists who want to standardise settings across their team - save a profile in the standalone pipeline, and it will be available for selection within the workflow wizard as well.

Note that saving the entire workflow configuration as a single profile is not currently supported. Profiles are applied per step.

For more information on saving and managing profiles, see Saving your analysis settings as profiles.
 

Viewing your results

This workflow will produce a Biologics Annotation Result document, which is the same document as running the NGS Antibody Annotator standalone pipeline. The result is saved in the same folder as your input files, or in the folder specified in the Output step.

Like any other Biologics Annotator Result document, you can also:

• Filter your Sequences
• View the Graphs for Quality Assurance and Graphs to interpret Clusters and Clonotypes
• Perform Sequence Alignments
• View the "Clusters" in your dataset
• Add new Clusters to your Results
• Subset your sequences and re-calculate clusters
• Add Assay Data to your Analysis Results
• Compare Results across Multiple Experiments to monitor clonal expansion or to identify sequences present across multiple datasets.
• Edit your Sequences
• Repair low-quality sequences after Annotation