ScalePlex Analysis

ScalePlex analysis involves processing pooled samples and assigning cells back to their original fixation plate wells. This section covers the analysis workflows, parameters, and data processing steps.

Input Requirements

Workflow Configuration

Required Parameter:

# In runParams.yml or command line
scalePlex: true

Sample Barcode Table Additions

There are additional optional columns for the Sample Barcode Table when running the ScalePlex pipeline.

Column	Description	Example
scalePlexLibIndex2	(Quantum Only) Index PCR sequences to associate with enriched library. Full sequence list here: ScalePlex i5 Quantum	QSR-1;QSR-2
scalePlexLibIndex	(Scale RNA v1.1 Only) i7 sequences to associate with enriched library. Full sequence list here: ScalePlex i7	ScalePlex-A-AP1
scalePlexBarcodes	Valid fixation plate wells for this sample, follows same specification scheme as the RNA RT barcodes, but is in reference to the ScalePlex fixation plate	1A-6H

View example sample barcode tables (including for ScalePlex).

Tip

non-ScalePlex: "Sample" = RT plate well groups (e.g., Donor1 in wells 1A-3H)
ScalePlex: "Sample" = RT plate well containing a pool of samples
The pipeline processes the pool as one sample, then assigns cells back to original samples meaning users original biological sample may differ from the pipeline "sample" definition

Sample Pool Processing

How Analysis By Pool Works

For pipeline inputs and outputs, a "sample" (which is a required entry in the sample barcode table) represents the pool of biological samples fixed in one ScalePlex fixation. This pool is loaded into specific RT wells and treated as a single "sample" for processing and reporting, though it typically contains multiple biological samples. When you define a "sample" in the sample barcode table, you are defining the RT barcode pool for which those ScalePlex samples were loaded. The pipeline reports "sample"-level metrics based on this pool and performs cell calling. Once cell calling is complete on the "sample"-level data, the ScalePlex workflow begins and assigns ScalePlex oligos, allowing cells to be traced back to their origin on the ScalePlex fixation plate. When viewing the Sample Report for a RT barcode pool, the Summary tab shows a single Barcode Rank Plot and single values for Read Metrics and Cell Metrics for the pool. As described here, the ScalePlex tab provides detailed information about the ScalePlex library performance, deconvolution into individual ScalePlex-fixed samples, the number of successfully assigned cells, and associated sensitivity metrics.

Analysis Report Structure

Summary Tab:

Single Barcode Rank Plot for the entire pool
Combined Read Metrics and Cell Metrics
Pool-level statistics

ScalePlex Tab:

Detailed deconvolution performance
Individual sample assignment success rates
Sensitivity metrics for each original sample

Configuration

Analysis Parameters

Cell Assignment Methods

ScalePlex can use one of two two algorithms for assigning cells back to their original fixation plate wells. The choice between these methods depends on your experimental design and the diversity of ScalePlex oligos in your pool. This is controlled by the --scalePlexAssignmentMethod parameter in the workflow.

Background-Based Assignment (Default: `bg`)

The background-based method is the default approach for ScalePlex assignment. This method estimates a background profile for each ScalePlex oligo across all cells in the pool, then performs statistical testing to identify significantly enriched oligos in each cell. The algorithm validates that the enriched oligo combinations match the expected ScalePlex plate layout, where each well is uniquely identified by a pair of oligos (one from a row and one from a column).

When to Use: This method will run by default unless otherwise specified, and should work well for most experiments.

Fold Change Assignment (`fc`)

This method compares the fold change between the second and third highest detected ScalePlex oligo counts in each cell, rather than comparing against a global background estimate. If they do pass the fold change threshold, then the top two oligos are validated as above for the bg method for the expected pairing.

When to Use: This is the recommended method whenever there is low ScalePlex oligo diversity in your pool or lower overall ScalePlex oligo counts per cell. Specifically, use this method when:

Your ScalePlex pool consists of samples from only a single row OR a single column of the fixation plate in some cases
You have low ScalePlex oligo counts per cell
The background method fails to assign cells properly
Top ScalePlex Fraction plot looks ideally distributed but assignment remains low

Note If users want to change the assignment method but not have to rerun everything else in the workflow, this can be accomplished using a reporting run, see Reporting

Quality Control Parameters

Enrichment Threshold (`scalePlexPercentFromTopTwo`)

Controls the percentage of ScalePlex UMIs that must come from the top two oligos for a valid assignment. Set to 0 by default (disabled), but can be increased to 50-80 for stricter quality control.

Fold Change Threshold (`scalePlexFCThreshold`)

When using the fold change method, this parameter sets the minimum fold change required between the second and third highest oligo counts. The default value is 2, but can be increased to 3-5 for stricter assignment criteria.

ScalePlex Library

When using the ScalePlex Oligo Fixation Plate a separate, paired enriched library is generated from each index PCR reaction to allow further demultiplexing of cells. Additional analysis options to process the reads from this library are detailed here.

BCL Input Mode

Requirements:

ScaleRNA and ScalePlex libraries sequenced together on same flowcell
scalePlex: true parameter enabled
Sample barcode table configured for RNA library

Processing Steps:

Detection: Workflow detects potential ScalePlex library indices
Demultiplexing: Separates RNA and ScalePlex reads
Sample Splitting: Divides data by RT well designation
Parallel Analysis: Processes both library types simultaneously

FASTQ Input Mode

When starting with fastq files or if the ScaleRNA and ScalePlex libraries were sequenced separately, fastq files will need to be generated for ScaleRNA and ScalePlex into separate files, then placed in one parent directory supplied in the fastqDir parameter (files can exist in subdirectories of this supplied path). See Fastq Generation for more information and view example samplesheets.

Requirements:

Both RNA and ScalePlex FASTQ files must be in the same directory or have a common parent directory
--fastqDir parameter pointing to that directory
Sample barcode table configured for RNA library

File Organization:

fastq_directory/
├── Sample1_R1_001.fastq.gz
├── Sample1_R2_001.fastq.gz
├── Sample1_I1_001.fastq.gz
├── Sample1_I2_001.fastq.gz
├── Sample1-SCALEPLEX_R1_001.fastq.gz
├── Sample1-SCALEPLEX_R2_001.fastq.gz
├── Sample1-SCALEPLEX_I1_001.fastq.gz
├── Sample1-SCALEPLEX_I2_001.fastq.gz
...

Data Processing Workflow

RNA Processing

Standard Workflow:

Cell calling performed on entire pool
Gene expression quantification
Quality metrics calculation

Additional Trimming:

ScalePlex PCR Handles: Removed to prevent contamination
Purpose: Eliminate ScalePlex molecules from RNA fraction
Benefit: Cleaner gene expression data

ScalePlex Processing

Parallel Analysis:

Barcode Validation: Same validation as RNA workflow
Oligo Detection: Validates ScalePlex target sequences
UMI Counting: Generates oligo-specific count matrices
Quality Assessment: Per-oligo quality metrics

Cell Assignment Process

Step 1: Background Estimation (bg method):

Calculate expected background for each oligo across all cells
Statistical testing against background
Identify significantly enriched oligos

OR

Step 1: Fold Change Analysis (fc method):

Compare 2nd vs 3rd highest oligo counts
Apply fold change threshold
Identify cells with clear oligo enrichment

Step 2: Combination Validation:

Verify oligo pairs match expected plate layout
Check against user-specified scalePlexBarcodes
Validate row + column combinations

Step 3: Assignment:

Assign cells to original fixation plate wells
Generate assignment confidence scores
Flag assignment failures

Quality Control

Assignment Success Metrics

Key Indicators:

Assignment Rate: Percentage of cells successfully assigned
Top ScalePlex Fraction: Proportion from top two oligos
Saturation: UMI detection efficiency

Troubleshooting

Low Assignment Rates:

Check ScalePlex oligo quality and concentration
Verify fixation protocol
Review assignment parameters

High Background:

Adjust scalePlexPercentFromTopTwo threshold
Consider using fold change method
Check for oligo contamination

Unexpected Combinations:

Verify ScalePlex plate layout
Check oligo design and sequences
Review sample pooling process

ScalePlex Overview - General ScalePlex information
ScalePlex Outputs - Output files and formats
Sample Barcode Table - Configuration requirements
QC Reports - Quality control metrics

Need Help?

For more information, please contact support@scale.bio or visit our support website.