Question: Why do I need a custom recipe when sequencing Multiome ATAC libraries on the NextSeq?
Answer: The NextSeq 500/550 runs a reverse complement sequencing workflow. Therefore you would need to follow the sequencing recommendations for reverse complement strand sequencing workflows:
However, the NextSeq 500/550 software does not support an i5 indexing read of 24bp. In order to accurately capture and read the 10x barcode, the use of a custom sequencing recipe is required. A custom sequencing recipe that specifies the following requirements is necessary for sequencing:
The custom sequencing recipe specifies 8 dark cycles (to skip sequencing of the 8bp spacer), and 16 cycles read (to capture 10x barcode).
Make sure to install a custom sequencing recipe file before sequencing Multiome ATAC libraries on the NextSeq500/550. It not installed, and just 16 bps are read without a custom sequencing recipe: The 8bp Spacer is captured along with only 8bp of the 10x barcode. An 8bp 10x barcode is too short to correctly assign transposed fragments to individual nuclei. Libraries sequenced without a custom recipe on the NextSeq 500/550 will need to be resequenced with the correct configurations.
More details on expected data outputs and metrics of Multiome ATAC libraries can be found in the Sequencing Metrics & Base Composition of Single Cell Multiome ATAC Libraries Technical Note
Note: A custom sequencing recipe is not needed for sequencing standalone ATAC (v1, v1.1) libraries.
Products: Single Cell Multiome ATAC + Gene Expression