When sequencing 10X Multiome ATAC libraries on the NextSeq 500/550, a custom recipe is required to account for the 24 bp i5 index. This bulletin shows how to install the custom recipe and how to set up runs using the custom recipe. For more information why a custom recipe is needed for 10X Multiome ATAC libraries, see this article on the 10X Genomics website.    

To obtain the custom recipe, email techsupport@illumina.com and request a custom recipe for 10X Multiome ATAC libraries for NextSeq 500/550.

Confirm which NextSeq Control Software (NCS) version is installed on the instrument. From the NCS home screen, select Manage Instrument and then select About to determine the NCS version.

After obtaining the custom recipe from Illumina Tech Support, add the custom recipe folders to your instrument.

Using the custom recipe with NCS v4

Installing the custom recipe

1. Unzip the custom recipe folders obtained by Tech Support
2. Copy each unzipped folder to the appropriate location on your NextSeq

• The folder 'NSQ8DCI2v2.5_MO' is the Mid Output folder, and it should be placed in the 'Mid' folder, located here:
  • C:\Program Files\Illumina\NextSeq Control Software\Recipe\Custom\Mid
• The folder 'NSQ8DCI2v2.5_HO' is the High Output folder, and it should be placed in the 'High' folder, located here:
  • C:\Program Files\Illumina\NextSeq Control Software\Recipe\Custom\High

Figure 1: Folder structure for installing the custom recipe in NCS v4

Setting up a run with the custom recipe in NCS v4

1. Go to Manage Instrument from the home screen, select System Customization, select the Enable Custom Recipes checkbox, and select Save.
2. To use the custom recipe, the NextSeq must be run in standalone mode. Local Run Manager (LRM) and BaseSpace do not support custom recipes.
3. Run Setup
   1. Choose Manual as run mode, do not select BaseSpace for storage.
   2. Under Run Setup, enter the desired read length (50 x 8 x 16 x 49).
   3. Load the flow cell.
   4. Load the buffer.
   5. Load the reagents.
   6. On the bottom right of the next window, you will see a Recipe drop-down. It is set to 'High' by default. Select the drop-down and select the custom recipe for your sequencing kit (Figure 1).
   7. Select Next and proceed with run set up as usual.

Figure 2. Selecting a custom recipe in NCS v4 during run set up.

After sequencing is complete, change all settings back to proceed with original standard run recipes. 

Select Manage Instrument from the home screen, select System Customization, deselect the Enable Custom Recipes checkbox, and select Save. Standard recipes will be usable with this option enabled.

Using the custom recipe with NCS v2

Installing the custom recipe

1. Unzip the Customer recipe folders obtained by Tech Support
2. Copy each unzipped folder to the appropriate location on your NextSeq

• The folder 'NSQ8DCI2v2.5_MO' is the Mid Output folder, and it should be placed in the 'Mid' folder, located here:
  • C:\Illumina\NextSeq Control Software\Recipe\Custom\Mid

• The folder 'NSQ8DCI2v2.5_HO' is the High Output folder, and should be placed in the 'High' folder, located here:
  • C:\Illumina\NextSeq Control Software\Recipe\Custom\High

Setting up a run with the custom recipe in NCS v2

1. Place instrument in standalone mode.
   To use the custom recipe, the NextSeq must be run in standalone mode. BaseSpace does not support custom recipes.
2. To put the instrument in standalone mode, select Manage Instrument from the home screen, select System Configuration. Click on the BaseSpace configuration option, then select the Standalone
3. Run Set up
1. Under Run Set Up, enter the Run Name and Library ID.
2. Select the Custom Recipe from the Recipe drop-down (Figure 2).
3. Choose Paired End for the Read Type.
4. Enter the desired read length (50 x 8 x 16 x 49).
5. Select Next and proceed with run set up as usual.




Figure 3. Setting up a run with a custom recipe in NCS v2.

After sequencing is complete, change all settings back to proceed with original standard run recipes. Select Manage Instrument from the home screen, select System Configuration. Click on the BaseSpace configuration option, then select the BaseSpace option.