Prepare and Add Custom Primers
Custom primers are diluted using HT1, and then added to the custom wells in the NextSeq 1000/2000 reagent cartridge. Make sure that the reagent cartridge is thawed and inspected before proceeding.
If using custom or third-party libraries alongside PhiX or Illumina libraries, prepare your custom read
primers by spiking into VP21 and custom index primers by spiking into VP14.
HT1, VP21, and VP14 are included in the NextSeq 1000/2000 primer kits. Each kit configuration includes different reagents. HT1 is not included in the NextSeq 1000/2000 sequencing reagent kit and is offered as a standalone reagent for purchase. To verify the appropriate kit option for your libraries, refer to Kit Configurations.
Prepare Custom Read Primers
For most custom library types, the read primers are pooled together in one custom primer well and the index primers are pooled together in the other custom primer well. It is not recommended to combine a read primer with an index primer because of volume constraints.
In the following steps, the volume of custom primer used depends on the custom primer stock concentration.
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1.
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If frozen, thaw each custom primer to be used. |
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2.
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If using custom or third-party libraries only, prepare custom read primers and/or custom index primers as follows. |
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Use HT1 to dilute the custom read primer mix to yield a total volume of 600 µl with each custom read primer at 0.3 µM final concentration. |
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Use HT1 to dilute the custom index primer mix to yield a total volume of 600 µl with each custom index primer at 0.6 µM final concentration. |
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If using custom or third-party libraries alongside PhiX or Illumina libraries, prepare custom read primers or custom index primers as follows. |
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Add each custom read primer mix to 600 μl HP21 for a 0.3 μM final concentration. |
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Add each custom index primer mix to 600 μl BP14 for a 0.6 μM final concentration. |
Add Custom Primers to the Reagent Cartridge
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1.
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Using a clean pipette tip, pierce the foil seal covering the custom 1 well or custom 2 well as needed. |
- Well 1
- Well 2
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2.
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Add 550 μl custom primer mix to custom 1 well or custom 2 well on the reagent cartridge. If using both wells, add 550 μl custom primer mix to the second well. |
Dispense the liquid slowly to avoid spillage, bubbles, and cross-contamination.
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3.
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Initiate the sequencing run within 1 hour. Storing the cartridge inside the system can cause the custom primer mix to evaporate. |
