Spiking custom primers into the Illumina sequencing primers for MiSeq i100 Series

Background

Libraries prepared with custom sequencing primers require optimization to perform successfully on Illumina sequencing platforms. Combining (or “spiking”) custom primers into the Illumina primers is necessary when including PhiX or other standard Illumina libraries with the custom sample.

Illumina cannot guarantee the performance or compatibility of custom primers, and it is up to the customer to validate primers for sequencing on a specific platform.

Important Note: Custom primers are not supported in Index First workflow.

General Overview

• Prepare and add the appropriate volume of each custom primer, or custom primer mix, to the custom primer well on the dry cartridge.

• Configure the options in the Review Run screen to use the custom primers:

  • De-select Sequence Indexes First

  • Select the appropriate checkboxes for the intended custom primers.

All other steps follow the typical run setup workflow. Refer to Set Up a Run for Custom Primers, and then proceed to Protocol for sequencing protocol instructions.

Prepare Custom Primers

1. If frozen, thaw each custom primer to be used.

2. If using custom or third-party libraries with PhiX or Illumina libraries, prepare custom read primers or custom index primers as follows:

   1. Add each custom read primer mix to 500 μl VP21 for a 0.3 μM final concentration.

   2. Add each custom index primer mix to 500 μl VP14 for a 0.6 μM final concentration.

Part number of Read and/or Index Primer kits (NextSeq 1000/2000 custom primer kit configurations are compatible with the MiSeq i100 Series):

NextSeq 1000/2000 XLEAP-SBS Read & Index Primer Kit: Part Number 20112856

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NextSeq 1000/2000 XLEAP-SBS Index Primer Kit: Part Number 20112858

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NextSeq 1000/2000 XLEAP-SBS Read Primer Kit: Part Number 20112859

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Add Custom Primers to the Dry Cartridge

Once prepared, refer to Dry Cartridge for well locations below for adding the custom primers to the cartridge:

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1. Using a clean pipette tip, pierce the foil seal covering the appropriate custom primer well on the dry cartridge.

2. Add 500 μl custom primer to the appropriate well.

*Dispense the liquid slowly to avoid spillage, bubbles, and cross-contamination.

For any feedback or questions regarding this article (Illumina Knowledge Article #9551), contact Illumina Technical Support [e-mail].