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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. 1.
    Thaw the SB2 in a room temperature water bath for 60 minutes.
  2. 2.
    Thaw the FDR and HP10 reagents in a room temperature water bath for 20 minutes.
  3. 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)
  1. 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
  1. 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.
  2. 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
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