Custom primer use workflow for iSeq 100 control software version 3.0

Before starting To use custom sequencing primers on the iSeq 100, the custom primer option must be enabled in the control software, and the run must be performed in Manual Mode. See the iSeq Product Documentation Custom Primers section for more information on this and the subsequent steps below.

The following is the process for use of custom sequencing primers.

• Note: if PhiX will also be included in the library pool, use Illumina read sequencing primers to dilute the custom read sequencing primers.

Dilute custom primers

• Use HT1 or HP21 (Illumina insert read primer mix) to dilute each custom read primer mix to yield 140 µl at 0.3 µM final concentration.

• Use HT1 or BP14 (Illumina index read primer mix) to dilute each custom index primer mix to yield 140 µl at 0.6 µM final concentration.

Initiate run set up

1. Select Sequence in the iSeq 100 Control Software run setup.

2. Select Yes to verify the run uses a custom primer.

3. Load the cartridge and the flow cell, and then select Close door. Do not add the library at this time.

4. In the Run Setup page, select one of the following options for each read and index:

• No—Use existing Illumina primer in the reagent cartridge. No is the default selection.

• Yes—Use the custom primer.

5. Select Start Pre-Run Checks.

• Once pre-run checks begin, the run parameters cannot be changed, and the consumables cannot be reused.

• Wait about 15 minutes for pre-run checks to complete.

Add custom primers to the regent cartridge

1. Using a new pipette tip, pierce the library reservoir and push the foil to the edges to enlarge the hole.

• Discard the pipette tip to prevent contamination.

2. Add 140 μl custom primer to the bottom of the reservoir on the reagent cartridge. Avoid touching the foil.

3. Load the cartridge, select Close Door, and then Yes, Close Door.

• Custom primer loading can take about 8 minutes per custom primer.

4. If more than one custom primer was selected in Run Setup, repeat steps 2-3.

5. When prompted, add 20 μl diluted library to the bottom of the reservoir on the reagent cartridge. Avoid touching the foil.

6. Load the cartridge, select Close Door & Start Run, and then Yes, Close Door to initiate sequencing.

• Initiate sequencing within 1 hour of preparing the custom primer. Storing the cartridge inside the system can cause the custom primer mix to evaporate.

Notes:

• HT1 is sold separately from the iSeq 100 sequencing consumables.

• Illumina sequencing primers are available for purchase separately. See the Accessory Products section on this page.

If sequencing Illumina DNA PCR Free libraries, see Custom primer requirements for the Illumina DNA PCR-Free Prep, Tagmentation kit.

For any feedback or questions regarding this article (Illumina Knowledge Article #6250), contact Illumina Technical Support [e-mail].