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# Demultiplexing FASTQs with bcl2fastq

Though cellranger-atac mkfastq is the preferred option for converting BCLs to FASTQs compatible with Cell Ranger ATAC FASTQs, there are some cases where you need to use bcl2fastq from Illumina® to generate FASTQs. This will be necessary if you do not wish to demultiplex an entire flow cell at once. You may also choose this method if bcl2fastq is more tightly integrated into your sequencing workflow.

Demultiplexing Chromium data with bcl2fastq from Illumina® requires the correct specification of the sample sheet and command-line options. This guide will walk you through what you'll need to do to generate Cell Ranger ATAC-compatible FASTQs.

## Sample sheet generator

You will need to create a sample sheet in order to get bcl2fastq to correctly embed the names of samples into output FASTQ files. There is a key difference to keep in mind when creating sample sheets for a Chromium run. Each Chromium sample index set is actually a blend of four different sequence oligos, and each oligo must be represented as a separate row in the sample sheet. This means that for every sample being demultiplexed from the flow cell, there should be 4 lines in the sample sheet.

The tool below will help you accurately generate data lines for your sample sheet. When you plan an experiment, you should know the name of the sample index set used for each sample, which comes from the reagent kit (such as "SI-P01-A2"). For each sample, enter its lane, sample name, and sample index set below, and then press 'Add'. When you're done, you can either copy and paste comma-separated output directly into a text editor to create a sample sheet CSV, or copy/paste tab-separated output into a spreadsheet such as Microsoft Excel:

Comma (CSV) Tab (for Spreadsheet copy)

If you are just running a single sample in a lane, then you can have a single line with the index blank, though bcl2fastq will include reads associated with any sample index.

## Running bcl2fastq

Illumina® bcl2fastq must be called with the correct --use-bases-mask argument and other arguments in order to properly demultiplex and output FASTQs for all the reads in a Chromium library. In the examples below, ${FLOWCELL_DIR} is the directory that contains a flow cell's Data folder, ${OUTPUT_DIR} is the directory that you want to output FASTQs to, and ${SAMPLE_SHEET_PATH} is the path to the sample sheet CSV you created. bcl2fastq Version 2.20 or higher $ bcl2fastq --use-bases-mask=Y50,I8,Y16,Y49 \
--ignore-missing-positions \
--ignore-missing-controls \
--ignore-missing-filter \
--ignore-missing-bcls \
-r 6 -w 6 \
-R ${FLOWCELL_DIR} \ --output-dir=${OUTPUT_DIR} \
--interop-dir=${INTEROP_DIR} \ --sample-sheet=${SAMPLE_SHEET_PATH}


In both cases, if you want to limit bcl2fastq to a subset of lanes, you will need to supply values to the --tiles argument.

### Omitting extra bases from reads

If you add extra bases to a sample index read, you will need to account for this in the --use-bases-mask argument. For example, if you ran a sample index read with 9 bases, you will need to truncate the last base in order for Cell Ranger ATAC to run correctly.

You can exclude a single base by adding a single n character to the read argument, or adding n* to exclude all bases after a certain position. See below:

Single Cell ATAC chemistry

i5 Index Read (I2)1617Y16n
After generating FASTQs, you should be able to follow the pipeline instructions. The value of --sample should be the name of the sample, which can be found in the Sample_Name column in your sample sheet. The value of --fastqs should be ${OUTPUT_DIR}/${PROJECT_NAME} where ${OUTPUT_DIR} is as defined above and ${PROJECT_NAME} is the value in the Sample_Project column in the sample sheet.