# 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](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/dna-pcr-free-prep.html).

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](https://support.illumina.com/downloads/illumina-dna-pcr-free-reference-guide.html) 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](https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-reference_material-list/000003343) 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](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-dna-pcr-free-loading-concentration-tech-note-770-2020-007/illumina-dna-pcr-free-loading-concentration-tech-note-770-2020-007.pdf). 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](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-dna-prep-pcr-free-index-correction-tech-note-m-gl-00005/illumina-dna-pcr-free-index-correction-tech-note-m-gl-00005.pdf) 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.

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