Considerations before starting the Illumina DNA PCR Free library preparation

What is required for library prep and sequencing? Does this library prep workflow need anything out of the ordinary?

Library preparation kit, index primer kit, additional consumables and equipment, and sequencing reagents are required.

For library preparation and index kit offerings, see the Illumina DNA PCR-Free Prep product page.

For some sequencers, custom sequencing primers are also required.

• The NovaSeq 6000 1.5 reagent kits and NovaSeq X / X Plus reagent kits include all the necessary primers for sequencing Illumina DNA PCR-Free libraries; no additional sequencing primers are required.

• For all other supported Illumina sequencing instruments:

  • Illumina DNA PCR-Free Prep Sequencing Primers Read 1 + Index 2 (catalog 20041797) (contains VP10 and VP14 primer mixtures).

  • Note: catalog 20041796 (contains VP10 primer mix only) has been obsolesced, with last order date of October 30, 2023.

See equipment and consumables sections of the Reference Guide before beginning, for a comprehensive list of additional requirements.

What important considerations are there for each sequencing instrument?

If using an instrument that requires custom sequencing primers, see above, and also see the Custom primer requirements for the Illumina DNA PCR Free Prep, Tagmentation kit article for additional information on custom primer usage and run set up considerations. Failure to use custom primers when required will result in sequencing failure.

For loading concentration information, see Optimal loading concentrations for Illumina DNA PCR-Free libraries. Loading concentrations may need to be further optimized in each lab to optimize run performance and data output.

To mitigate variability in index representation with the IDT for Illumina DNA/RNA UD Indexes, see the Balancing sample coverage for whole-genome sequencing technical note.

What are the typical applications for Illumina DNA PCR-Free?

Using genomic DNA (gDNA), whole blood, dry blood spots (DBS), or saliva for whole-genome sequencing (WGS).

Will Illumina PCR-Free libraries be normalized at the end of the workflow?

Some workflows will. The BLT-PF tagmentation bead reagent will normalize assay yield at or above 300 ng DNA input for the gDNA workflow. The integrated extraction workflow uses a lower BLT-PF volume, and consequently normalizes at 200 ng.

Can Illumina DNA PCR-free libraries be used for downstream enrichment capture?

No, Illumina DNA PCR-Free libraries are not compatible with downstream enrichment.

Can Illumina DNA PCR-Free libraries be PCR amplified?

This is not recommended.

For any feedback or questions regarding this article (Illumina Knowledge Article #3430), contact Illumina Technical Support [e-mail].