Question: I have sequenced single cell ATAC or multiome ATAC libraries with a NextSeq 1000/2000 or NovaSeq X series instrument. When I use bcl2fastq to demultiplex, I am not able to generate FASTQ files. Or the FASTQ files that I generate are not able to work with Cell Ranger ARC or Cell Ranger ATAC. What should I do?
Answer: BCL Convert is planned to replace bcl2fastq in the future. Illumina recommends using BCL Convert for demultiplexing NextSeq 1000/2000 and NovaSeq X/X Plus runs to generate FASTQ files for single cell ATAC and multiome ATAC. The older bcl2fastq software has not been updated to be fully compatible with these instruments. BCL Convert is available to run on-board NextSeq 1000/2000 and NovaSeq X/X Plus instruments, in BaseSpace, or on the Linux command line.
There is an issue that occurs because of the sample sheet formatting when runs from these instruments are demultiplexed on bcl2fastq. You may see reads going into only the Undetermined files or there may be reads containing all Ns.
For instructions on how to run BCL Convert and to obtain example sample sheets with appropriate settings for generating FASTQ files for single cell/multiome ATAC libraries, please see the following pages on the 10x Genomics Support website:
The following article has instructions on how to setup demultiplexing on BaseSpace or use BaseSpace to create and download a sample sheet CSV:
Related articles:
- Upgrading from bcl2fastq to BCL Convert (Illumina)
- NovaSeq X Series sample sheet settings (Illumina)
- Does mkfastq work with NovaSeq X data? (10x Genomics)