Where are your FASTQ files?

• In an output folder from mkfastq or bcl2fastq (fastq_path) and:

  • Scenario 1: In a subdirectory next to Reports and Stats folders, with expected sample name prefixes.
  • Scenario 2: Within multiple folders per sample index, like "SI-NA-A1_1" and "SI-NA-A1_2".
  • Scenario 3: In the same directory as the Reports and Stats folders.

• In a different folder:

  • Scenario 4: 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).
• Scenario 5: Like "read-I1-AAAAAAA_lane-001-chunk-001.fastq.gz".
• Scenario 6: Unlike any of the above examples.

How did I get here?

By running cellranger-atac mkfastq, or by running bcl2fastq directly (with an IEM samplesheet) on a flow cell. If you ran mkfastq, your files will be in a (MKFASTQ_ID)/outs/fastq_path folder, and your file hierarchy probably looks something like this:

Copy
MKFASTQ_ID
|-- MAKE_FASTQS_CS
`-- outs
    |-- fastq_path
        |-- HFLC5BBXX
            |-- test_sample1
            |   |-- test_sample1_S1_L001_I1_001.fastq.gz
            |   |-- test_sample1_S1_L001_R1_001.fastq.gz
            |   |-- test_sample1_S1_L001_R2_001.fastq.gz
            |   |-- test_sample1_S1_L001_R3_001.fastq.gz
            |   |-- test_sample1_S1_L002_I1_001.fastq.gz
            |   |-- test_sample1_S1_L002_R1_001.fastq.gz
            |   |-- test_sample1_S1_L002_R2_001.fastq.gz
            |   |-- test_sample1_S1_L002_R3_001.fastq.gz
            |   |-- test_sample1_S1_L003_I1_001.fastq.gz
            |   |-- test_sample1_S1_L003_R1_001.fastq.gz
            |   |-- test_sample1_S1_L003_R2_001.fastq.gz
            |   `-- test_sample1_S1_L003_R3_001.fastq.gz
            |-- test_sample2
            |   |-- test_sample2_S2_L001_I1_001.fastq.gz
            |   |-- test_sample2_S2_L001_R1_001.fastq.gz
            |   |-- test_sample2_S2_L001_R2_001.fastq.gz
            |   |-- test_sample2_S2_L001_R3_001.fastq.gz
            |   |-- test_sample2_S2_L002_I1_001.fastq.gz
            |   |-- test_sample2_S2_L002_R1_001.fastq.gz
            |   |-- test_sample2_S2_L002_R2_001.fastq.gz
            |   |-- test_sample2_S2_L002_R3_001.fastq.gz
            |   |-- test_sample2_S2_L003_I1_001.fastq.gz
            |   |-- test_sample2_S2_L003_R1_001.fastq.gz
            |   |-- test_sample2_S2_L003_R2_001.fastq.gz
            |   `-- test_sample2_S2_L003_R3_001.fastq.gz
        |-- Reports
        |-- Stats
        |-- Undetermined_S0_L001_I1_001.fastq.gz
        ...
        `-- Undetermined_S0_L003_R2_001.fastq.gz

If you ran bcl2fastq directly, then the output root folder would be 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 sample sheet. Other situations described later on this page deal with the presence of four separate sets of files (four "samples" from bcl2fastq's point of view) per single biological sample/library.

For more information on the naming conventions, please visit the Illumina® 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 would pass into any analysis pipeline to target the right FASTQ files in this scenario. Be sure to substitute the capitalized text as appropriate. Also note that in most cases you will be passing a single sample into any given pipeline. Exceptions to this are described in the documentation for the individual pipelines. The "All Samples" entries in this table are provided for technical completeness.

How did I get here?

It is likely that an input sample sheet was used that explicitly separated the four oligos in a 10x Genomics sample index set into four separate sample names. You may see a file hierarchy like this:

Copy
bcl2fastq_output
|-- HFLC5BBXX
    |-- SI-GA-A1_1
    |   |-- SI-GA-A1_1_S1_L001_I1_001.fastq.gz
    |   |-- SI-GA-A1_1_S1_L001_R1_001.fastq.gz
    |   |-- SI-GA-A1_1_S1_L001_R2_001.fastq.gz
    |   `-- SI-GA-A1_1_S1_L001_R3_001.fastq.gz
    |-- SI-GA-A1_2
    |   |-- SI-GA-A1_2_S2_L001_I1_001.fastq.gz
    |   |-- SI-GA-A1_2_S2_L001_R1_001.fastq.gz
    |   |-- SI-GA-A1_2_S2_L001_R2_001.fastq.gz
    |   `-- SI-GA-A1_2_S2_L001_R3_001.fastq.gz
    |-- SI-GA-A1_3
    |   |-- SI-GA-A1_3_S3_L001_I1_001.fastq.gz
    |   |-- SI-GA-A1_3_S3_L001_R1_001.fastq.gz
    |   |-- SI-GA-A1_3_S3_L001_R2_001.fastq.gz
    |   `-- SI-GA-A1_3_S3_L001_R3_001.fastq.gz
    |-- SI-GA-A1_4
    |   |-- SI-GA-A1_4_S4_L001_I1_001.fastq.gz
    |   |-- SI-GA-A1_4_S4_L001_R1_001.fastq.gz
    |   |-- SI-GA-A1_4_S4_L001_R2_001.fastq.gz
    |   `-- SI-GA-A1_4_S4_L001_R3_001.fastq.gz
|-- Reports
|-- Stats
|-- Undetermined_S0_L001_I1_001.fastq.gz
|-- Undetermined_S0_L001_R1_001.fastq.gz
|-- Undetermined_S0_L001_R2_001.fastq.gz
`-- Undetermined_S0_L001_R3_001.fastq.gz

You probably want to be able to merge All samples from the SI-GA-A1 index into a single analysis. If you only run one index at a time, you will see a smaller number of reads than expected, which may translate to lower coverage or cell count than you expect for your experiment.

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 likely looks something like this:

Copy
fastq_path
|-- Reports
|-- Stats
|-- test_sample_S1_L001_I1_001.fastq.gz
|-- test_sample_S1_L001_R1_001.fastq.gz
|-- test_sample_S1_L001_R2_001.fastq.gz
|-- test_sample_S1_L001_R3_001.fastq.gz
|-- test_sample_S1_L002_I1_001.fastq.gz
|-- test_sample_S1_L002_R1_001.fastq.gz
|-- test_sample_S1_L002_R2_001.fastq.gz
|-- test_sample_S1_L002_R3_001.fastq.gz
|-- test_sample_S1_L003_I1_001.fastq.gz
|-- test_sample_S1_L003_R1_001.fastq.gz
|-- test_sample_S1_L003_R2_001.fastq.gz
|-- test_sample_S1_L003_R3_001.fastq.gz
|-- Undetermined_S0_L001_I1_001.fastq.gz
...
`-- Undetermined_S0_L003_R2_001.fastq.gz

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

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 may look like this:

Copy
PROJECT_FOLDER
|-- MySample_S1_L001_I1_001.fastq.gz
|-- MySample_S1_L001_R1_001.fastq.gz
|-- MySample_S1_L001_R2_001.fastq.gz
|-- MySample_S1_L001_R3_001.fastq.gz
|-- MySample_S1_L002_I1_001.fastq.gz
|-- MySample_S1_L002_R1_001.fastq.gz
|-- MySample_S1_L002_R2_001.fastq.gz
|-- MySample_S1_L002_R3_001.fastq.gz

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

How did I get here?

The 10x Genomics demux pipeline was used to demultiplex the flow cell instead of mkfastq. This pipeline has been deprecated, but you have still got a job to do. Your file hierarchy likely has many files in it, named as such:

Copy
demux_id
|-- BCL_PROCESSOR_CS
`-- outs
    |-- fastq_path
        |-- read-I1_si-AAAAAAAA_lane-001-chunk-001.fastq.gz
        ...
        |-- read-I1_si-TTTTTTTT_lane-002-chunk-001.fastq.gz
        |-- read-I1_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 will need to know the 10x sample index or oligos associated with your sample. That will select the correct files from the sample indices in your folder:

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 Genomics pipelines need files named in the bcl2fastq or demux convention in order to run properly. You will 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 flow cell.

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

[Sample Name]S1_L00[Lane Number][Read Type]_001.fastq.gz

Where Read Type is one of:

• I1: Dual index i7 read (optional)
• R1: Read 1
• R2: Dual index i5 read
• R3: Read 2

Alternatively, Cell Ranger ATAC will also accept ATAC FASTQs in this format:

• I1: Dual index i7 read (optional)
• R1: Read 1
• I2: Dual index i5 read
• R2: Read 2

After you have renamed those files into one of those formats, use the following arguments: