Custom primer requirements for the Illumina DNA PCR Free Prep, Tagmentation kit

Illumina DNA PCR-Free Prep, Tagmentation libraries require custom sequencing primers for successful sequencing on certain Illumina platforms because these libraries use novel chemistry. Failure to use the specified custom sequencing primers for these libraries when required results in sequencing failure. This article reviews the custom sequencing primer kit contents, custom primer loading volume and position requirements per instrument, and how to set up runs using custom sequencing primers.

The custom sequencing primers, sold separately, are provided at final concentration and require no further dilution. They are loaded into the dedicated custom primer wells, rather than spiked into the Illumina primer wells. For full instructions, refer to the Custom Primers Guide or Product Documentation for the relevant instrument, listed in the Resources section of this article.

These custom sequencing primers are compatible with Illumina DNA PCR-Free libraries, PhiX control library (Cat. No. FC-110-3001), and all currently supported/compatible Illumina libraries.

The required custom sequencing primers are sold separately from the Illumina DNA PCR-Free library preparation kit.

Illumina DNA PCR-Free Sequencing and Index Primer (Cat. No. 20041797)

• Contains VP10 Custom Read 1 Primer (2 tubes X 7.5 ml) and VP14 Custom Index 2 Primer (2 tubes X 10.5 ml).

• Note that the VP14 Custom Index 2 Primer is not used on all instruments.

Custom primer loading volume and position requirements per instrument:

a. Requires iSeq 100 Control Software v3.0 or higher. Custom primers workflow must be enabled and primers added sequentially. See iSeq 100 Sequencing System Guide for further instructions. b. Libraries can be sequenced with either Reads First or Index First workflow. c. Requires NextSeq 1000/2000 Control Software v1.2 or higher and v3 flow cells.

Run set up instructions per software:

* See the MiniSeq (MNCS v2)/NextSeq 500/550 (NCS v4) Local Run Manager library prep kit definition file for Illumina DNA PCR-Free libraries with IDT for Illumina DNA/RNA UD Indexes or for Illumina DNA/RNA UD Indexes and steps to import as custom library. ** MiSeq (MCS > v3.0) Local Run Manager library prep kit definition file for Illumina DNA PCR-Free libraries with IDT for Illumina DNA/RNA UD Indexes or for Illumina DNA/RNA UD Indexes and steps to import as custom library. a. Leave "Denature and Dilute Onboard" box checked. For more details on appropriate well selection for NextSeq 1000/2000, please see the NextSeq 1000 and 2000 Custom Primers Guide. b. Read length is limited by the number of SBS cycles available in reagent kits, maximum read length for Illumina sequencing platforms, and 10 bp dual indexes. See the following for additional information. * How many cycles of SBS chemistry are in my kit?

• Maximum read length for Illumina sequencing platforms

c. IEM v1 sample sheet creation is required only for MiSeq run setup on control software 2.6 or lower.

Resources: NovaSeq Series Custom Primers Guide NextSeq 500 and NextSeq 550 Sequencing Systems Custom Primers Guide NextSeq 1000/2000 Custom Primers Guide

MiSeq System Custom Primers Guide MiniSeq System Custom Primers Guide iSeq 100 Sequencing System Guide Optimal loading concentrations for Illumina DNA PCR-Free libraries application note

For any feedback or questions regarding this article (Illumina Knowledge Article #3343), contact Illumina Technical Support [e-mail].