Prepare and Add Custom Primers

Custom primers are diluted using HT1, and then added to the custom primer wells in the NextSeq 1000/2000 reagent cartridge. Make sure that the reagent cartridge is thawed and inspected before proceeding.

To prepare custom or third-party libraries alongside PhiX or Illumina libraries, spike custom read primers and custom index primers into the appropriate reagent as follows.

• XLEAP-SBS
– Custom read primers: VP21
– Custom index primers: VP14
• Standard SBS
– Custom read primers: BP14
– Custom index primers: HP21

For most custom library types, you can pool the read primers can be pooled together in one custom primer well and the index primers can be pooled together in the other custom primer well. For optimal base calling performance, avoid combiningdo not combine a custom read primer with a custom index primer.

In the following steps, the volume of custom primer used depends on the custom primer stock concentration.

1. If frozen, thaw each custom primer to be used.
2. If using custom or third-party libraries only, prepare custom read primers and/or custom index primers as follows.
• 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.
• 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.
3. If using custom or third-party libraries alongside PhiX or Illumina libraries, prepare custom read primers or custom index primers as follows.
• Add each custom read primer mix to 600 μl VP21 or HP21 for a 0.3 μM final concentration.
• Add each custom index primer mix to 600 μl VP14 or BP14 for a 0.6 μM final concentration.
1. Using a clean pipette tip, pierce the foil seal covering the custom 1 well or custom 2 well as needed.

  1. Custom 1 Well
  2. Custom 2 Well
2. 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.

3. Initiate the sequencing run within 1 hour. Storing the cartridge inside the system can cause the custom primer mix to evaporate.