Cell Ranger1.3, printed on 12/26/2024
The cellranger count command provides the means to define samples according to the output of a single cellranger mkfastq 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.
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.
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-GRCh38-1.2.0/GRCh38", 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:
Make a copy of this _invocation file; this will be the MRO from which we will build our multi-flowcell invocaction MRO.
Continuing with the example MRO above, we would make the following changes:
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 "sc_rna_counter_cs.mro" call SC_RNA_COUNTER_CS( sample_id = "sample345-multi", sample_def = [ { "fastq_mode": "ILMN_BCL2FASTQ", "gem_group": null, "lanes": null, "read_path": "/home/jdoe/runs/HAWT7ADXX", "sample_indices": [ "any" ], "sample_names": [ "Sample1" ] }, { "fastq_mode": "ILMN_BCL2FASTQ", "gem_group": null, "lanes": null, "read_path": "/home/jdoe/runs/HAWPUADXX", "sample_indices": [ "any" ], "sample_names": [ "Sample1" ] } ], sample_desc = "", reference_path = "/opt/refdata-cellranger-GRCh38-1.2.0/GRCh38", 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
The officially supported method of doing this is to run cellranger count on each library as per the single-library instructions above, then combine the results using the more efficient cellranger aggr pipeline as covered here. The method described below is included only for backwards compatibility.
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 "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-GRCh38-1.2.0/GRCh38", recovered_cells = null, )
The cellular barcode sequences will include suffixes from the different gem groups.
AGAATGGTCTGCAT-1 GTAGCAACGTCGTA-2
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.
| A MRO ParseError: unexpected token error suggests that your sample_def entry is not valid JSON. Ensure that your commas are (and are not) present 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 - 2.1.2 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