Cell Ranger1.0, printed on 11/21/2024
| The demux pipeline is not supported by current versions of cellranger. Please refer to the most recent version of cellranger documentation for help running demultiplexing software (mkfastq or Illumina's BCL software) to convert BCL files to FASTQ file format for analysis with cellranger pipelines. |
The cellranger demux pipeline is the first step in analyzing a Chromium sequencer run. It takes an Illumina BCL output folder and demultiplexes based on the 8bp sample index read, and generates FASTQs for the R1 and R2 paired-end reads as well as the sample index.
While this pipeline runs Illumina bcl2fastq as one its stages, it produces a FASTQ output folder whose layout is better optimized for parallelized analysis than the standard file layout produced by bcl2fastq alone.
For the following example, it is assumed that you have already installed:
Cell Ranger package such that cellranger demux --help returns without errors.
Illumina's bcl2fastq such that either bcl2fastq --version (v2.17 and higher) or configureBclToFastq.pl --help (v1.8.4) returns without errors.
The cellranger demux command requires only the path to a BCL sequencer output folder:
$ cellranger demux --run=/sequencing/140101_D00123_0111_AHAWT7ADXX cellranger demux Copyright (c) 2016 10x Genomics, Inc. All rights reserved. ----------------------------------------------------------------------------- Martian Runtime - 1.3.0 Running preflight checks (please wait)...
(Martian is 10x Genomics' pipeline execution framework.)
cellranger demux will first run "preflight checks" to ensure that there are no critical errors with the arguments you provided or in your environment settings. Following the preflight checks, the runtime will begin running pipeline stages:
Running preflight checks (please wait)... 2016-05-01 12:00:00 [runtime] (ready) ID.HAWT7ADXX.BCL_PROCESSOR_CS.BCL_PROCESSOR.ANALYZE_RUN 2016-05-01 12:00:00 [runtime] (ready) ID.HAWT7ADXX.BCL_PROCESSOR_CS.BCL_PROCESSOR.BARCODE_AWARE_BCL2FASTQ 2016-05-01 12:00:03 [runtime] (split_complete) ID.HAWT7ADXX.BCL_PROCESSOR_CS.BCL_PROCESSOR.ANALYZE_RUN 2016-05-01 12:00:03 [runtime] (run:local) ID.HAWT7ADXX.BCL_PROCESSOR_CS.BCL_PROCESSOR.ANALYZE_RUN.fork0.chnk0.main ...
If you encounter any preflight errors, please refer to the Troubleshooting page.
Once the cellranger demux pipeline has successfully completed, the output can be found in a new folder named with the serial number of the flowcell processed by cellranger demux. The flowcell serial number in this example is HAWT7ADXX:
$ ls -l drwxr-xr-x 4 jdoe jdoe 4096 May 1 13:39 HAWT7ADXX
The demultiplexed FASTQ files can be found in outs/fastq_path:
$ ls -l HAWT7ADXX/outs/fastq_path/ -rw-r--r-- 1 jdoe jdoe 3071801 May 1 13:39 read-I1_si-AAACGTAC_lane-001-chunk-000.fastq.gz ... -rw-r--r-- 1 jdoe jdoe 52246181 May 1 13:39 read-RA_si-GTGGAATT_lane-001-chunk-000.fastq.gz -rw-r--r-- 1 jdoe jdoe 3759265 May 1 13:39 read-RA_si-X_lane-001-chunk-000.fastq.gz
It is important not to change the naming of these FASTQ files, as the cellranger pipeline depends on the specific file structure produced by cellranger demux. The layout of the pipestance output folder is described in more detail in the Pipestance Structure section.