Comment on page
How to perform a Read 1 rehybridization of a HiSeq v4 high output flow cell on the cBot

Rehybridization of a HiSeq v4 high output flow cell is typically performed on the HiSeq instrument, but in some cases, a Read 1 rehybridization can be performed on the cBot. This bulletin describes the steps and reagents necessary to perform this rehybridization.
The following reagents and consumables are needed:
FDR, SB2, and the Read 1 sequencing primer (HP10)** from a HiSeq Multi-Primer Rehyb v4 Kit v4 [GD-403-4001].
Leftover HT1 (> 3.6 ml) from the HiSeq v4 cluster kit.
The HiSeq v4 high output sequencing flow cell to be rehybridized.
A used HiSeq v4 cBot cluster plate with the original tube strips.*
*If a used HiSeq v4 clustering plate is not available, you can use 8-tube strips from a different cluster plate. The tube strips should be thoroughly cleaned with laboratory-grade water to avoid reagent contamination.
**If you are using custom primers, replace HP10 with 0.5 µM of your custom Read 1 sequencing primer.
Before starting, note the following:
At least 3.6 ml of HT1 from the cluster kit is needed to perform the rehybridization.
The HiSeq v4 rehybridization recipes are available on v3.0.46 or later of the cBot software.
Follow these steps to perform the rehybridization:
1.Thaw the SB2 in a room temperature water bath for 60 minutes.
2.Thaw the FDR and HP10 reagents in a room temperature water bath for 20 minutes.
3.Pipette the reagents into the tube strips in the following rows with the volumes indicated:
Row 1 - HT1 (at least 450 µl per tube)
Row 2 - HP10 (at least 150 µl per tube)
Row 5 - FDR (at least 250 µl per tube)
Row 7 - SB2 (at least 350 µl per tube)
4.Select the appropriate v4 rehybridization recipe based on the type of flow cell:
PE_HiSeq_Cluster_Kit_v4_cBot_Rehyb_recipe_v9.0
SR_HiSeq_Cluster_Kit_v4_cBot_Rehyb_recipe_v9.0
5.Load the flow cell and scan the used HiSeq v4 plate ID. If using a different type of reagent plate, manually input the reagent plate ID as GA1234567-PE4.
6.Load the cluster plate onto the cBot and follow the on-screen prompts to complete the run setup.
When the rehybridization is complete, use the rehybridized flow cell to set up a new run on the HiSeq.





For any feedback or questions regarding this article (Illumina Knowledge Article #1529), contact Illumina Technical Support [email protected].
Last modified 12d ago