10x Genomics
Chromium Single Cell Gene Expression

Cell Ranger1.0, printed on 12/21/2024

Running Multi-Flowcell Samples

The cellranger run command provides the means to define samples according to the output of a single cellranger demux command's output FASTQs. While this sample specification (i.e., one flowcell per sample) is the most common, high-depth sequencing often involves either sequencing a single sample or combining multiple samples across multiple flowcells. To this end, the cellranger pipeline allows complex sample construction such as combining sample indices from multiple flowcells into a single cellranger run.

To create complex sample specifications, you will have to write your own MRO file for the cellranger pipeline. MRO is the language used to define pipelines to the Martian pipeline framework which is responsible for managing pipeline execution. The cellranger command is simply a shell script that converts command line arguments into an MRO file which is passed to the Martian pipeline execution command, cellranger mrp, and writing MROs directly allows you to access the full range of options available for each pipeline.

Understanding the Pipeline Invocation MRO

The easiest way to write your own MRO is to start with the MRO from a previous pipeline. Assuming you have already run a single-flowcell sample (e.g., sample345) you will be using for your multi-flowcell sample, examine the _invocation file contained in its output directory:

$ cat sample345/_invocation
 
@include "cellranger_cs.mro"
 
call CELLRANGER_CS(
    sample_id = "sample345",
    sample_def = [
        {
            "gem_group": null,
            "lanes": null,
            "read_path": "/home/jdoe/runs/HBA2TADXX",
            "sample_indices": [ "any" ],
            "fastq_mode": "BCL_PROCESSOR",
            "bc_read_type": "I1",
            "si_read_type": "I2"
        }
    ],
    sample_desc = "",
    reference_path = "/opt/refdata-cellranger-1.0.0/hg19",
    recovered_cells = null,
)

The sample_def argument controls the parameters used to define this sample and is a JSON-encoded list of maps that define:

• read_path - a directory containing FASTQs from a single flowcell
• lanes - a list of lanes from this flowcell to be included in this sample (e.g., [ 1, 2 ], [ 2 ], etc) or null to use all lanes
• gem_group - indicates GEM chip channel corresponding to a single sample across multiple flowcells. This field will be described in more detail in the next section.
• fastq_mode - set this to "BCL_PROCESSOR"
• bc_read_type - set this to "I1"
• si_read_type - set this to "I2"
• sample_indices - a list of sample indices from the 8-bp I5 read

Make a copy of this _invocation file; this will be the MRO from which we will build our multi-flowcell invocation MRO.

Combining Multiple Flowcells

Continuing with the example MRO above, we would make the following changes:

1. Give this sample a new sample_id.
2. Duplicate the dict contained in the sample_def definition as a second item in the sample_def list
3. Change the read_paths for each of these sample_def objects to point to the HAWT7ADXX and HAWPUADXX FASTQ output directories
4. Change lanes and/or sample_indices to reflect the flowcell configuration used in sequencing, if necessary
5. Change gem_group to reflect single or multiple sample configuration across flowcells

Analyzing a Single Sample Across Multiple Flowcells

It may be useful to sequence a sample multiple times that was generated from the same GEM chip channel. To set up a single sample multi-flowcell cellranger run, the gem_group field in each sample_def argument must be set to null. cellranger will then set the gem group to 1 for all flowcells within the sample.

For example, the two-flowcell MRO may appear as

$ cp sample345/_invocation sample345-multi.mro
$ nano sample345-multi.mro
...
 
$ cat sample345-multi.mro
 
@include "cellranger_cs.mro"
 
call CELLRANGER_CS(
    fastq_mode = "BCL_PROCESSOR",
    sample_id = "sample345-multi",
    sample_def = [
        {
            "gem_group": null,
            "lanes": null,
            "read_path": "/home/jdoe/runs/HAWT7ADXX",
            "sample_indices": [ "any" ],
            "fastq_mode": "BCL_PROCESSOR",
            "bc_read_type": "I1",
            "si_read_type": "I2"
        },
        {
            "gem_group": null,
            "lanes": null,
            "read_path": "/home/jdoe/runs/HAWPUADXX",
            "sample_indices": [ "any" ],
            "fastq_mode": "BCL_PROCESSOR",
            "bc_read_type": "I1",
            "si_read_type": "I2"
        }
    ],
    sample_desc = "",
    reference_path = "/opt/refdata-cellranger-1.0.0/hg19",
    recovered_cells = null,
)

where the changes from the original MRO are highlighted.

All cellular barcode sequences produced in the BAM from cellranger will include a suffix 1.

AGAATGGTCTGCAT-1

Analyzing Multiple Samples Across Multiple Flowcells

It may be useful to combine multiple similar samples from separate GEM chip channels into a single cellranger run. To set up a multiple sample multi-flowcell cellranger run, the gem_group field in each sample_def argument must be set to incrementally increasing integers, starting with 1.

$ cp sample345/_invocation sample345-multi.mro
$ nano sample345-multi.mro
...
 
$ cat sample345-multi.mro
 
@include "cellranger_cs.mro"
 
call CELLRANGER_CS(
    fastq_mode = "BCL_PROCESSOR",
    sample_id = "sample345-multi",
    sample_def = [
        {
            "gem_group": 1,
            "lanes": null,
            "read_path": "/home/jdoe/runs/HAWT7ADXX",
            "sample_indices": [ "any" ],
            "fastq_mode": "BCL_PROCESSOR",
            "bc_read_type": "I1",
            "si_read_type": "I2"
        },
        {
            "gem_group": 2,
            "lanes": null,
            "read_path": "/home/jdoe/runs/HAWPUADXX",
            "sample_indices": [ "any" ],
            "fastq_mode": "BCL_PROCESSOR",
            "bc_read_type": "I1",
            "si_read_type": "I2"
        }
    ],
    sample_desc = "",
    reference_path = "/opt/refdata-cellranger-1.0.0/hg19",
    recovered_cells = null,
)

The cellular barcode sequences will include suffixes from the different gem groups.

AGAATGGTCTGCAT-1
GTAGCAACGTCGTA-2

Running Cell Ranger

Once you have this multi-flowcell MRO, confirm that its syntax is valid with cellranger mrc, the MRO compiler included with Cell Ranger:

$ cellranger mrc sample345-multi.mro
Successfully compiled 1 mro files.

Then run the MRO file using cellranger's alternate MRO-mode syntax:

$ cellranger run sample345-multi sample345-multi.mro --uiport=3600
Martian Runtime - 1.3.0
Serving UI at http://localhost:3600                                             
 
Running preflight checks (please wait)...
2012-01-01 12:00:00 [runtime] (ready)           ID.sample345-multi.PHASER_SVCALLER_CS.PHASER_SVCALLER._ALIGNER.SETUP_CHUNKS
2012-01-01 12:00:00 [runtime] (run:local)       ID.sample345-multi.PHASER_SVCALLER_CS.PHASER_SVCALLER._SNPINDEL_PHASER.SORT_GROUND_TRUTH
2012-01-01 12:00:00 [runtime] (run:local)       ID.sample345-multi.PHASER_SVCALLER_CS.PHASER_SVCALLER._SNPINDEL_PHASER.SORT_GROUND_TRUTH.fork0.chnk0.main

where

• The first argument (sample345-multi) is the name of the run/sample.
• The second argument (sample345-multi.mro) is the custom MRO file you just created.
• Additional optional parameters (e.g., --uiport=3600) can be added to the end of the cellranger command.