10x Genomics
Visium Spatial Gene Expression

Space Ranger2.0, printed on 12/26/2024

Specifying Input FASTQ Files for 10x Pipelines

The spaceranger count pipeline requires FASTQ files as input, which typically come from running spaceranger mkfastq, a 10x-aware convenience wrapper for bcl2fastq. However, it is possible to use FASTQ files from other sources, such as Illumina's bcl2fastq, a published dataset, or our bamtofastq.

For experiments with Gene Expression (GEX) data, here are the arguments available for specifying which FASTQ files spaceranger count should use:

Argument	Brief Description
--fastqs	Required for GEX analysis. The folder containing the FASTQ files to be analyzed. Generally, this is the fastq_path folder generated by spaceranger mkfastq. If the files are in multiple folders, for instance because one library was sequenced across multiple flow cells, supply a comma-separated list of paths.
--sample	Optional. Sample name to analyze. This is as specified in the sample sheet supplied to mkfastq or bcl2fastq. Multiple names may be supplied as a comma-separated list, in which case they are treated as one sample.
--lanes	Optional. Lanes associated with this sample. Defaults to all lanes.

FASTQ file naming convention

There is a wide range of ways bcl2fastq and mkfastq can be invoked, resulting in a wide range of potential file names and locations as the output.

To serve as input for spaceranger, FASTQ files should conform to the naming conventions of bcl2fastq and mkfastq:

[Sample Name]_S1_L00[Lane Number]_[Read Type]_001.fastq.gz

Where Read Type is one of:

• I1: Sample index read (optional)
• I2: Sample index read (optional)
• R1: Read 1
• R2: Read 2

The FASTQ files are specified by providing the path to the folder containing them (via the --fastqs argument) and then optionally restricting the selection by specifying the samples and or lanes of interest.

Finding the right FASTQ files to process and the right arguments to process those files as desired can be confusing. To assist users, this page illustrates examples of how to handle common scenarios involving different FASTQ file folder hierarchies or naming conventions.

Quick Start:

Where are your FASTQ files?

• In an output folder from mkfastq or bcl2fastq (fastq_path) and:
  • In a subdirectory next to Reports and Stats folders, with expected sample name prefixes.
  • Within multiple folders per sample index, like "SI-TT-A1_1" and "SI-TT-A1_2".
  • In the same directory as the Reports and Stats folders.
• In a different folder:
  • I don't see Reports or Stats anywhere. The files are named like "MySample_S1_L001_I1_001.fastq.gz"

How are they named?

• Consistent with bcl2fastq/mkfastq, e.g. "MySample_S1_L001_I1_001.fastq.gz" (see above).
• Like "read-I1-AAAAAAA_lane-001-chunk-001.fastq.gz".
• Unlike any of the above examples.

Scenario: My FASTQs are in an output folder from mkfastq or bcl2fastq, in a subdirectory next to Reports and Stats folders, with expected sample name prefixes

How did I get here?

By running mkfastq with a simple CSV layout file or Illumina Experiment Manager samplesheet, or by running bcl2fastq directly (with an IEM samplesheet) on a flowcell. If you ran mkfastq, your files are in a (MKFASTQ_ID)/outs/fastq_path folder, and your file hierarchy looks similar to this:

MKFASTQ_ID
|-- MAKE_FASTQS_CS
`-- outs
    |-- fastq_path
        |-- HFLC5BBXX
            |-- test_sample1
            |   |-- test_sample1_S1_L001_I1_001.fastq.gz
            |   |-- test_sample1_S1_L001_I2_001.fastq.gz
            |   |-- test_sample1_S1_L001_R1_001.fastq.gz
            |   |-- test_sample1_S1_L001_R2_001.fastq.gz
            |   |-- test_sample1_S1_L002_I1_001.fastq.gz
            |   |-- test_sample1_S1_L002_I2_001.fastq.gz
            |   |-- test_sample1_S1_L002_R1_001.fastq.gz
            |   |-- test_sample1_S1_L002_R2_001.fastq.gz
            |   |-- test_sample1_S1_L003_I1_001.fastq.gz
            |   |-- test_sample1_S1_L003_I2_001.fastq.gz
            |   |-- test_sample1_S1_L003_R1_001.fastq.gz
            |   `-- test_sample1_S1_L003_R2_001.fastq.gz
            |-- test_sample2
            |   |-- test_sample2_S2_L001_I1_001.fastq.gz
            |   |-- test_sample2_S2_L001_I2_001.fastq.gz
            |   |-- test_sample2_S2_L001_R1_001.fastq.gz
            |   |-- test_sample2_S2_L001_R2_001.fastq.gz
            |   |-- test_sample2_S2_L002_I1_001.fastq.gz
            |   |-- test_sample2_S2_L002_I2_001.fastq.gz
            |   |-- test_sample2_S2_L002_R1_001.fastq.gz
            |   |-- test_sample2_S2_L002_R2_001.fastq.gz
            |   |-- test_sample2_S2_L003_I1_001.fastq.gz
            |   |-- test_sample2_S2_L003_I2_001.fastq.gz
            |   |-- test_sample2_S2_L003_R1_001.fastq.gz
            |   `-- test_sample2_S2_L003_R2_001.fastq.gz
        |-- Reports
        |-- Stats
        |-- Undetermined_S0_L001_I1_001.fastq.gz
        |-- Undetermined_S0_L001_I2_001.fastq.gz
        ...
        `-- Undetermined_S0_L003_R2_001.fastq.gz

If you ran bcl2fastq directly, then the output root folder is where fastq_path is in the hierarchy above.

"Expected sample name prefixes" means you have one set of fastq files per sample, prefixed with the name of the sample as it appears in the simple CSV layout file or IEM samplesheet.

For more information on the naming conventions, visit Illumina's support site or refer to the bcl2fastq User Guide. The scenario where your files do not conform to the naming convention is described in a different section later on this page.

The table below describes the arguments you pass into the pipeline to target the right fastq files in this scenario. Be sure to substitute the capitalized text as appropriate.

Situation	Argument+Value
mkfastq	--fastqs=MKFASTQ_ID/outs/fastq_path
mkfastq, multiple flowcells	--fastqs=MKFASTQ_ID/outs/fastq_path1,MKFASTQ_ID/outs/fastq_path2
bcl2fastq direct	--fastqs=/PATH/TO/bcl2fastq_output
Process test_sample1 from all lanes (mkfastq)	--fastqs=MKFASTQ_ID/outs/fastq_path \ --sample=test_sample1
Process test_sample1 from lane 1 only (mkfastq)	--fastqs=MKFASTQ_ID/outs/fastq_path \ --sample=test_sample1 \ --lanes=1
Process test_sample1 and test_sample2 as a single merged sample (mkfastq)	--fastqs=MKFASTQ_ID/outs/fastq_path \ --sample=test_sample1,test_sample2

 

Scenario: My FASTQs are in an output folder from mkfastq or bcl2fastq, in the same directory as the Reports and Stats folders

How did I get here?

An Illumina Experiment Manager-formatted samplesheet was used with either no entry or a blank entry for the Sample_Project column. Your hierarchy looks similar to this:

fastq_path
|-- Reports
|-- Stats
|-- test_sample_S1_L001_I1_001.fastq.gz
|-- test_sample_S1_L001_I2_001.fastq.gz
|-- test_sample_S1_L001_R1_001.fastq.gz
|-- test_sample_S1_L001_R2_001.fastq.gz
|-- test_sample_S1_L002_I1_001.fastq.gz
|-- test_sample_S1_L002_I2_001.fastq.gz
|-- test_sample_S1_L002_R1_001.fastq.gz
|-- test_sample_S1_L002_R2_001.fastq.gz
|-- test_sample_S1_L003_I1_001.fastq.gz
|-- test_sample_S1_L003_I2_001.fastq.gz
|-- test_sample_S1_L003_R1_001.fastq.gz
|-- test_sample_S1_L003_R2_001.fastq.gz
|-- Undetermined_S0_L001_I1_001.fastq.gz
|-- Undetermined_S0_L001_I2_001.fastq.gz
...
`-- Undetermined_S0_L003_R2_001.fastq.gz

This is correct; use the same arguments as if the FASTQs were organized into subfolders within the output folder.

Situation	Argument+Value
mkfastq	--fastqs=MKFASTQ_ID/outs/fastq_path
bcl2fastq direct	--fastqs=/PATH/TO/bcl2fastq_output
Process test_sample from all lanes (mkfastq)	--fastqs=MKFASTQ_ID/outs/fastq_path \ --sample=test_sample
Process test_sample from lane 1 only (mkfastq)	--fastqs=MKFASTQ_ID/outs/fastq_path \ --sample=test_sample \ --lanes=1

 

Scenario: My FASTQs are in a different folder; I don't see Reports or Stats anywhere. The files are named like "MySample_S1_L001_I1_001.fastq.gz"

How did I get here?

It is likely that FASTQ files have been transferred from either a mkfastq or bcl2fastq run into another folder. They still retain the names assigned by bcl2fastq, which is a combination of sample name, sample order, lane, read type, and chunk. Your file hierarchy looks similar to this:

PROJECT_FOLDER
|-- MySample_S1_L001_I1_001.fastq.gz
|-- MySample_S1_L001_I2_001.fastq.gz
|-- MySample_S1_L001_R1_001.fastq.gz
|-- MySample_S1_L001_R2_001.fastq.gz
|-- MySample_S1_L002_I1_001.fastq.gz
|-- MySample_S1_L002_I2_001.fastq.gz
|-- MySample_S1_L002_R1_001.fastq.gz
|-- MySample_S1_L002_R2_001.fastq.gz

This is correct; since the files are named according to the bcl2fastq standard, use the same arguments as if the FASTQs were organized into a flowcell folder or mkfastq output folder.

Situation	Argument+Value
No filtering according to sample or lane	--fastqs=/PATH/TO/PROJECT_FOLDER
Process MySample from all lanes	--fastqs=/PATH/TO/PROJECT_FOLDER \ --sample=MySample
Process MySample from lane 1 only	--fastqs=/PATH/TO/PROJECT_FOLDER \ --sample=MySample \ --lanes=1

 

Scenario: My FASTQs are named like "read-I1-AAAAAAA_lane-001-chunk-001.fastq.gz"

How did I get here?

The 10x demux pipeline was used to demultiplex the flowcell instead of mkfastq. This pipeline has been deprecated, but its output can still be used to run spaceranger count. Your file hierarchy likely has many files in it, named as such:

demux_id
|-- BCL_PROCESSOR_CS
`-- outs
    |-- fastq_path
        |-- read-I1_si-AAAAAAAA_lane-001-chunk-001.fastq.gz
        |-- read-I2_si-AAAAAAAA_lane-001-chunk-001.fastq.gz
        ...
        |-- read-I1_si-TTTTTTTT_lane-002-chunk-001.fastq.gz
        |-- read-I2_si-TTTTTTTT_lane-002-chunk-001.fastq.gz
        |-- read-I1_si-X_lane-002-chunk-001.fastq.gz
        |-- read-I2_si-X_lane-002-chunk-001.fastq.gz
        |-- read-RA_si-AAAAAAAA_lane-001-chunk-001.fastq.gz
        ...
        |-- read-RA_si-TTTTTTTT_lane-002-chunk-001.fastq.gz
        |-- read-RA_si-X_lane-002-chunk-001.fastq.gz

To ingest the correct FASTQ files from a demux run, you need to know the 10x sample dual-index associated with your sample. That selects the correct files from the sample indices in your folder:

Situation	Argument+Value
No filtering according to sample or lane	--fastqs=/PATH/TO/PROJECT_FOLDER
Process sample associated with SI-TT-A1	--fastqs=/PATH/TO/PROJECT_FOLDER \ --indices=SI-TT-A1
Process sample associated with SI-TT-A1, lane 1 only	--fastqs=/PATH/TO/PROJECT_FOLDER \ --indices=SI-TT-A1 \ --lanes=1

 

Scenario: My FASTQs are not named like any of the above examples.

How did I get here?

It is likely that you received files that were processed through a proprietary LIMS system, which employs its own naming conventions.

10x pipelines need files named in the bcl2fastq or demux convention in order to run properly. You need to determine which file corresponds to which sample and which read type, likely by consulting your sequencing core or the individual who demultiplexed your flowcell.

It is highly likely that these files were initially processed with bcl2fastq, so you need to rename the files in the following format, once you track down their origin:

[Sample Name]_S1_L00[Lane Number]_[Read Type]_001.fastq.gz

Where Read Type is one of:

• I1: Sample index read (optional)
• I2: Sample index read (optional)
• R1: Read 1
• R2: Read 2

Notice that I1 and I2 are optional because it is possible to have datasets without index reads at all if sequencing was performed without sample multiplexing. After you have renamed those files into that format, use the following arguments:

Situation	Argument+Value
No filtering according to sample or lane	--fastqs=/PATH/TO/PROJECT_FOLDER
Process SAMPLENAME from all lanes	--fastqs=/PATH/TO/PROJECT_FOLDER \ --sample=SAMPLENAME
Process SAMPLENAME from lane 1 only	--fastqs=/PATH/TO/PROJECT_FOLDER \ --sample=SAMPLENAME \ --lanes=1