10x Genomics Support/Cell Ranger/Tutorials/

Generating FASTQs

Converting BCL files to FASTQ format (demultiplexing) is a necessary step, especially when multiple libraries are pooled in one sequencing run. This article details three methods for generating FASTQ files from BCL files, all compatible with 10x Genomics Chromium libraries.

Software from Illumina, bcl2fastq and BCL Convert, may provide greater control over FASTQ generation parameters. Cell Ranger's mkfastq is a thin wrapper around Illumina's bcl2fastq.

Both bcl2fastq and BCL Convert are currently available from Illumina
Cell Ranger mkfastq

For inquiries about bcl2fastq or BCL Convert, contact Illumina Support at [email protected].

All three demultiplexing software methods require an input sample sheet and corresponding indices. The sample index sets are either dual index or single index.

Download the dual index kit sample index reference files at these links:

Download the single index kit sample index reference files at these links:

When preparing single index sample sheets for a Chromium run, remember that each index set comprises four distinct sequence oligos. Therefore, each oligo requires its own row, meaning the sheet should have four lines for every sample being demultiplexed.

After generating FASTQs, you are ready to run the cellranger count, cellranger vdj, or cellranger multi pipelines, depending on your data type and experimental design. Visit the Choosing a Pipeline page to find the Cell Ranger pipeline relevant to your data.