Setting Sample Sections in Cell Ranger Multi with Barcode-Sample-Assignment
Cell Ranger Multi allows you to analyze multiple samples simultaneously, a significant advantage for high-throughput single-cell RNA sequencing (scRNA-seq) experiments. A crucial step in this process is correctly assigning barcodes to their respective samples using a barcode-sample-assignment file. This file dictates how Cell Ranger Multi groups reads and assigns them to individual samples for downstream analysis. Let's break down how to set up this crucial element for successful multi-sample analysis.
What is a Barcode-Sample-Assignment File?
This file is a simple text file, typically a CSV (Comma Separated Values) or tab-separated file (TSV), that links each barcode sequence to a specific sample identifier. This link is essential because your sequencing run will generate reads containing barcodes, but without this file, Cell Ranger Multi wouldn't know which sample each barcode belongs to. The file usually has two columns:
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barcode: This column lists the unique barcode sequences used in your experiment to identify individual cells. These are the barcodes that Cell Ranger will look for in your sequencing data.
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sample: This column contains the name you've given to each sample. This name will then become the identifier for that sample's data in your final Cell Ranger output. Choose descriptive names to easily distinguish your samples later.
How to Create the Barcode-Sample-Assignment File
Creating the file depends on how your experiment was set up. There are a couple of key scenarios:
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Unique Barcodes per Sample: If each sample uses a completely unique set of barcodes, the file is straightforward. For each barcode in your experiment, list it along with the corresponding sample name.
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Shared Barcodes: If you have multiple samples that use some of the same barcodes (a common scenario with multiplexed experiments), you must carefully assign each barcode to only one sample. This requires a deep understanding of your experimental design and your barcode generation process. Inaccurate assignments can lead to significant errors in your analysis.
Example (CSV):
barcode,sample
AGTCGATC,SampleA
TGACGTCA,SampleB
CGATCGTA,SampleA
TAGCTAGC,SampleC
Example (TSV):
barcode sample
AGTCGATC SampleA
TGACGTCA SampleB
CGATCGTA SampleA
TAGCTAGC SampleC
How to Use the Barcode-Sample-Assignment File in Cell Ranger Multi
Once you have created your barcode-sample-assignment file (let's call it barcode_sample_assignment.csv), you run Cell Ranger Multi using the --sample-barcodes flag. This flag points Cell Ranger to the location of your file.
The command would look something like this:
cellranger multi --id=multisample_run --transcriptome=refdata-gex --fastqs=fastqs.csv --sample-barcodes=barcode_sample_assignment.csv
Replace the following:
multisample_run: Your chosen run ID.refdata-gex: The path to your Cell Ranger reference transcriptome.fastqs.csv: The path to your FASTQ file CSV (as generated by Cell Ranger).barcode_sample_assignment.csv: The path to your barcode-sample-assignment file.
Important Note: Ensure that the barcode sequences in your assignment file precisely match those present in your sequencing data. Any discrepancies will result in errors or misassignments.
Troubleshooting
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Incorrect Barcode Assignments: Double-check your barcode-sample-assignment file meticulously. Errors here lead to incorrect cell assignments and skewed results.
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Missing Barcodes: Verify that all barcodes identified in your sequencing data are present in the assignment file. Missing barcodes will result in the loss of data.
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File Formatting: Carefully adhere to the CSV or TSV format. Incorrect formatting will lead to Cell Ranger not recognizing the file. Use a text editor or spreadsheet program that allows saving in these formats.
By carefully constructing and correctly using the barcode-sample-assignment file, you ensure accurate and reliable results when performing multi-sample analysis with Cell Ranger Multi. Remember, accurate data entry is paramount for reliable downstream analyses.
