Software  ›   pipelines
If your question is not answered here, please email us at:  support@10xgenomics.com

10x Genomics
Chromium Single Cell Gene Expression

Generating FASTQs with cellranger demux

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.

If your sequencing run includes extra bases in barcode or sample index reads, or you only want to demultiplex part of your flowcell, you will need to follow the instructions on Generating FASTQs with bcl2fastq.

 

For the following example, it is assumed that you have already installed:

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 - 2.0.0
 
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.