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Sample Barcode Table

The sample barcode table (samples.csv) lists the samples included in an analysis run and any optional sample-specific analysis parameters.

The workflow processes all RT wells assigned to the same sample together, producing combined output for each. Importantly in the case of ScalePlex these "RT Samples" represents multiple biological samples pooled together after ScalePlex before the RT plate (RT sample pool). In the large or extra-large QuantumScale kits, a sample will be present across multiple sub-libraries (index PCR reactions). Each sub-library is initially analyzed separately, but samples are merged across sub-libraries for final output.

  • RT Sample (Pool): A group of RT plate wells (e.g., 1A-1H) that were loaded with the same input sample or ScalePlex sample pool. This is what the workflow considers a "sample" for processing and output generation.
  • ScalePlex / Biological Sample: The actual experimental samples indexed during ScalePlex Fixation (e.g., individual donors, treatment conditions, time points); See below and ScalePlex for details.
  • QuantumScale Sub-Library: The product of one PCR reaction, indexed by one of the QuantumScale PCR index primer pools; QSR-P for the small and medium kits; QSR1-8 for large and XL. These barcodes are sequenced in the Index2 (i5) read and given in the Sample Barcode Table as libIndex2.

File Format

The sample table is a comma-separated file (CSV) with a header line followed by one sample (or ScalePlex pool) per line. Example sample barcode tables for all QuantumScale configurations are available in quantum-sample-barcode-tables.

Basic format:

sample,barcodes,libIndex2
Donor1,1A-6H,QSR-1;QSR-2
Donor2,7A-12H,QSR-1;QSR-2

Required Columns

Column Description Example
sample Sample name (required) PBMC-2
barcodes RT plate wells for this sample (required for multi-sample experiments) 1A-2H

Naming Rules

  • Sample names can only contain letters, numbers and dashes (-)
  • Underscores are not valid
  • Must start with a letter

Optional Columns

Column Description Example Kit Type
libIndex2 PCR indices (semicolon-separated) QSR-1;QSR-2 QuantumScale
scalePlexLibIndex2 ScalePlex library index 2 QSR-1-SCALEPLEX QuantumScale - ScalePlex
scalePlexBarcodes ScalePlex barcodes 1A-6H ScalePlex
UTC Manual cell threshold per sample 500 All kits
libIndex Library index RNA-A-AP1 Only for ScaleRNA v1.1
scalePlexLibIndex ScalePlex library index ScalePlex-A-AP1 ScaleRNA v1.1 - ScalePlex
  • See ScalePlex Analysis for details on ScalePlex Sample Tables
  • libIndex2: If omitted, workflow searches for all possible PCR indices in input data
  • libIndex: Use for ScaleRNA v1.1 kit data
  • UTC: Enables manual cell thresholding per sample (see Cell Calling)

RT Barcode Format

RT barcodes are specified with the RT plate well in one of three formats:

  1. Individual Well: 1A
  2. Range of Wells: 1A-2H
  3. List of Values or Ranges: 1A;2A-2D

Important: Ranges are read in column-wise order:

  • 1A-2C refers to: 1A-1H (all of column 1) + 2A, 2B, 2C
  • Wells are sorted first by number (column), then by letter (row)
  • Use semicolon (;) to separate multiple wells or ranges

Row-Based Plating

For samples plated by rows, input each RT well separated by semicolons:

Row 1: 1A;2A;3A;4A;5A;6A;7A;8A;9A;10A;11A;12A
Row 4: 1D;2D;3D;4D;5D;6D;7D;8D;9D;10D;11D;12D

ScalePlex / RT pooling Examples

Example 1: ScalePlex Experiment (Multiple Biological Samples per RT Pool)

In a ScalePlex experiment, you can load multiple biological samples into the same RT barcode pool:

RT Plate Column 1 (1A-1H) = RT Barcode Pool "Sample1"

  • Donor1 (ScalePlex barcode A1)
  • Donor2 (ScalePlex barcode A2)
  • Donor3 (ScalePlex barcode A3)
  • ... (up to 96 donors in one RT pool)

Workflow Output: One BAM file named "Sample1" containing all 96 donors, with ScalePlex barcodes in the read tags for downstream demultiplexing.

Biological Reality: 96 separate biological samples that need post-processing to separate by ScalePlex barcodes. Please see ScalePlex Outputs for detailed information on demultiplexing ScalePlex samples.

Example 2: Standard RNA Kit (One Biological Sample per RT Pool)

In a standard RNA experiment without ScalePlex, each RT barcode pool typically represents one biological sample:

RT Plate Column 1 (1A-1H) = RT Barcode Pool "Donor1" = 1 Biological Sample

RT Plate Column 2 (2A-2H) = RT Barcode Pool "Donor2" = 1 Biological Sample

Workflow Output: Two separate BAM files - "Donor1.bam" and "Donor2.bam"

Example 3: Mixed Experimental Design

You might have a complex experiment with different sample types:

RT Plate Column 1 (1A-1H) = RT Barcode Pool "Control_Pool"

  • Control_Donor1 (ScalePlex A1)
  • Control_Donor2 (ScalePlex A2)
  • Control_Donor3 (ScalePlex A3)

RT Plate Column 2 (2A-2H) = RT Barcode Pool "Treatment_Pool"

  • Treatment_Donor1 (ScalePlex B1)
  • Treatment_Donor2 (ScalePlex B2)
  • Treatment_Donor3 (ScalePlex B3)

Workflow Output: Two BAM files - "Control_Pool.bam" and "Treatment_Pool.bam", each containing 3 biological samples that need ScalePlex demultiplexing.

Key Takeaway: Always consider your experimental design when interpreting workflow outputs. The workflow processes at the RT barcode pool level, but your analysis may require additional steps to separate biological samples within each pool.

ScaleRNA v1 Examples

ScaleRNA 3L v1.1 Kit

Rules for ScaleRNA 3L v1.1: - Use libIndex column instead of libIndex2 - barcodes is required if more than one sample is processed on the RT Barcode Plate - Follow the same barcode format rules as above

Example:

sample,barcodes,libIndex
Sample1,1A-6H,RNA-A-AP1
Sample2,7A-12H,RNA-A-AP1

Extended Throughput (ET) Plates

When using Extended Throughput plates, each ET plate has the same set of i5 barcodes but different i7 barcodes. The workflow expects pre-split fastq files based on i7 barcodes.

Required columns for ET: - sample: Sample name - barcodes: RT plate wells - libName (for fastq input) or libIndex (for bcl runFolder input)

Example with two ET plates:

sample,barcodes,libName
S1,1A-3H,ScaleRNA-A-AP1
S2,4A-6H,ScaleRNA-A-AP1
S3,7A-9H,ScaleRNA-A-AP1
S4,10A-12H,ScaleRNA-A-AP1
S1,1A-3H,ScaleRNA-A-AP2
S2,4A-6H,ScaleRNA-A-AP2
S3,7A-9H,ScaleRNA-A-AP2
S4,10A-12H,ScaleRNA-A-AP2

Note: The workflow combines data at the sample level while keeping single-cell barcodes separated during analysis.

How Sample Demultiplexing Works

Initial Barcode Parsing

  1. Extraction: Pipeline extracts single-cell barcodes from reads using ScaleBio's bc_parser tool
  2. Error Correction: Performed against expected barcode sequences
  3. Mismatch Tolerance: Allows up to 1 mismatch in barcode matching

Sample Demultiplexing

  1. RT Barcode Definition: Uses samples.csv to define RT barcodes for each sample
  2. Sample Separation: Separates different samples based on their RT plate well positions
  3. Output Generation: Creates an unaligned BAM file for each sample with cell barcodes in tags

Need Help?

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