Cell Ranger3.1, printed on 11/21/2024
As mentioned in the workflow overview, Cell Ranger can be used to analyze multiple GEM wells. Depending on your exact scenario, the approach may be different. Here are three circumstances, from most to least common:
You created one GEM well, but sequenced it more than once. This might have been to increase coverage depth, or for some other reason. You might have sequenced the GEM well across different lanes of the same flowcell, or across multiple flow cells. In any of these cases, provided that this all comes from a single GEM well, you can analyze these runs as a single sample using cellranger count by Specifying Input FASTQs.
You created GEM wells from multiple samples. This would be the case in many experiments, such as comparing diseased and healthy patients. First, run cellranger count on each GEM well separately. Then, to compare the GEM wells , aggregate them with cellranger aggr.
You created multiple GEM wells from the same sample. It is recommended to simply aggregate the GEM wells, just as if they were different samples. However, if it is necessary to pool these GEM gouprs and analyze them as a single sample, you can do that. It just requires learning a little more about exactly how Cell Ranger works, and writing a customized pipeline configuration file, called an MRO. This page covers this final scenario.
Cell Ranger uses a pipeline management framework called Martian. The pipeline and each stage in it is specified by a configuration file called an MRO file. Usually, the cellranger commands create the appropriate MRO files for you, but in the case that you want to do something outside the normal workflows, it is possible to create a custom MRO file to directly exercise the full range of features.
In the example below we describe how to construct an MRO file to specify multiple libraries as well as multiple flow cells (since most often the multiple libraries will have been sequenced on different runs).
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), examine the _invocation file contained in its output directory.
Note: this example assumes that the input flowcells were processed with cellranger mkfastq.
$ cat sample345/_invocation @include "sc_rna_counter_cs.mro" call SC_RNA_COUNTER_CS( sample_id = "sample345", sample_def = [ { "fastq_mode": "ILMN_BCL2FASTQ", "gem_group": null, "lanes": null, "read_path": "/home/jdoe/runs/HBA2TADXX", "sample_indices": [ "any" ], "sample_names": [ "Sample1" ] } ], sample_desc = "", reference_path = "/opt/refdata-cellranger-GRCh38-3.0.0", recovered_cells = null, force_cells = null, no_secondary_analysis = false, )
The sample_def argument controls the parameters used to define this sample and is a JSON-encoded list of maps that define:
Make a copy of this _invocation file; this will be the MRO from which we will build our multi-GEM well invocation MRO.
Continuing with the example MRO above, we would make the following changes:
$ cp sample345/_invocation sample345-multi.mro $ nano sample345-multi.mro ... $ cat sample345-multi.mro @include "sc_rna_counter_cs.mro" call SC_RNA_COUNTER_CS( sample_id = "sample345-multi", sample_def = [ { "fastq_mode": "ILMN_BCL2FASTQ", "gem_group": 1, "lanes": null, "read_path": "/home/jdoe/runs/HAWT7ADXX", "sample_indices": [ "any" ], "sample_names": [ "Sample1" ] }, { "fastq_mode": "ILMN_BCL2FASTQ", "gem_group": 2, "lanes": null, "read_path": "/home/jdoe/runs/HAWPUADXX", "sample_indices": [ "any" ], "sample_names": [ "Sample1" ] } ], sample_desc = "", reference_path = "/opt/refdata-cellranger-GRCh38-3.0.0", recovered_cells = null, force_cells = null, no_secondary_analysis = false, )
The cellular barcode sequences will include suffixes from the different GEM wells.
AGAATGGTCTGCAT-1 CTGATCGATATCGA-1 GTAGCAACGTCGTA-2 AGAATGGTCTGCAT-2
This is how cellranger prevents the same barcode from different cells in different GEM reactions from being erroneously combined into a single cell based only on the barcode sequence.
Once you have this single-sample, multi-GEM well, 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.
| A MRO ParseError: unexpected token error suggests that your sample_def entry is not valid JSON. Ensure that your commas are present if required (and absent if required) after each item. |
Then run the MRO file using cellranger's alternate MRO-mode syntax:
$ cellranger count sample345-multi sample345-multi.mro --uiport=3600 Martian Runtime - v3.2.3 Serving UI at http://localhost:3600 Running preflight checks (please wait)... 2016-11-14 17:26:40 [runtime] (ready) ID.patient123.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.SETUP_CHUNKS 2016-11-14 17:26:43 [runtime] (split_complete) ID.patient123.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.SETUP_CHUNKS 2016-11-14 17:26:43 [runtime] (run:local) ID.patient123.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.SETUP_CHUNKS.fork0.chnk0.main
where
