Setting Sample Sections in Cell Ranger Multi with Barcode-Sample-Assignment
Cell Ranger Multi allows you to process multiple samples simultaneously, greatly streamlining single-cell RNA sequencing (scRNA-seq) data analysis. A crucial step is correctly assigning barcodes to their respective samples using a barcode-sample-assignment file. This file dictates how Cell Ranger Multi organizes and analyzes your data, separating reads from each sample for independent processing. Let's explore how to create and use this file effectively.
What is a barcode-sample-assignment file?
The barcode-sample-assignment file is a crucial input for Cell Ranger Multi. It's a simple tab-separated file (TSV) that maps unique molecular identifiers (UMIs) or barcodes to specific samples. This is essential because multiple samples might be sequenced in a single lane, and this file enables Cell Ranger Multi to correctly demultiplex the data. Without it, Cell Ranger Multi won't know which reads belong to which sample, leading to incorrect results.
How to create a barcode-sample-assignment file?
The structure of the file is straightforward. It requires at least two columns:
-
barcode: This column lists the unique barcodes (or UMIs) used in your experiment. These barcodes identify individual cells. Ensure you use the exact barcode sequences as they appear in your sequencing data. Any discrepancies will lead to misassignments.
-
sample_name: This column indicates the sample to which each barcode belongs. Use consistent and descriptive sample names throughout your project.
Example:
barcode sample_name
AAACCTGAGAAACGT-1 SampleA
AAACCTGAGACCTGT-1 SampleB
AAACCTGCAGTTGGA-1 SampleA
AAACCTGGTCAAGCA-1 SampleB
... ...
This example shows four barcodes, two assigned to SampleA and two to SampleB. You'll need to repeat this for all barcodes in your experiment, mapping each one to its corresponding sample. The number of barcodes per sample will vary depending on the sequencing depth and the number of cells in each sample.
How to use the barcode-sample-assignment file in Cell Ranger Multi?
The barcode-sample-assignment file is passed to Cell Ranger Multi using the --sample-barcodes flag. The command will look something like this:
cellranger multi --id=multi_sample_run --transcriptome=/path/to/refdata-gex-GRCh38-2020-A --fastqs=/path/to/fastqs --sample-barcodes=/path/to/barcode-sample-assignment.tsv
Replace the placeholders with your actual paths. Crucially, ensure the path to your barcode-sample-assignment.tsv file is correct. A single typo can render your entire analysis unusable.
Troubleshooting common issues
-
Incorrect barcode assignment: Double-check your
barcode-sample-assignmentfile for typos and ensure the barcodes perfectly match the sequences in your sequencing data. Even a single base-pair difference will lead to misassignments. -
Missing barcodes: If some barcodes are missing from your assignment file, Cell Ranger Multi will not process the corresponding reads. This will result in an incomplete analysis and potentially biased results.
-
File format errors: Ensure your file is a properly formatted TSV file with tabs separating the columns and no extra spaces.
-
Path issues: Verify that the path you specify for the
barcode-sample-assignment.tsvfile is accurate and accessible to the Cell Ranger Multi command.
By carefully creating and using the barcode-sample-assignment file, you can accurately separate your samples within Cell Ranger Multi and obtain reliable, meaningful results from your scRNA-seq experiment. Remember to always double-check your file before running Cell Ranger Multi to prevent errors and ensure the integrity of your data.
