Question: Why do I need a custom recipe when sequencing Multiome ATAC libraries on the NextSeq?
Answer: The sequencing recommendations for Multiome ATAC libraries are as follows :
However, the NextSeq 500/550 software does not support an i5 indexing read of 24bp. 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).
Be sure to install a custom sequencing recipe file before sequencing Multiome ATAC libraries on the NextSeq500/550. If not installed, 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 assign transposed fragments to individual nuclei correctly. 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.
Read more: I installed a custom sequencing recipe on my NextSeq; why can't I input 24 bp in the i5 indexing read?
Products: Single Cell Multiome ATAC + GEX