Space Ranger2.0, printed on 11/12/2024
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. |
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 1R2
: Read 2The 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.
Where are your FASTQ files?
How are they named?
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 |
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 |
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 |
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 |
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 1R2
: Read 2Notice 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 \ |