> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-faq-list/000003444.md).

# Denature and dilution FAQ for Illumina DNA PCR Free

**Which Denature & Dilute protocol should be followed for NovaSeq 6000 and for other supported instruments? If protocol A/standard protocol, is NaOH added twice? (Once during the library preparation protocol (HP3 reagent) and then again during the denature & dilute protocol)**

Use the standard denature and dilution protocol (using NaOH) for instruments where denaturation is performed before loading the library on the instrument. Use the onboard denaturation protocol for instruments that support onboard denature and dilute.

While NaOH is used during the library preparation, it is still required to perform NaOH denaturation prior to sequencing to prevent the formation of any secondary structures.

**Do libraries need to be heat shocked/heat treated to make sure they are single stranded?**\
No. NaOH denaturation will resolve any secondary structures. Additional heat denaturation is not required.

**If denaturation prior to clustering was omitted**, **will the data be okay? What is the performance impact?**\
This method of clustering is not supported. However, because Illumina DNA PCR-free generates single stranded DNA (ssDNA) libraries, the libraries may still cluster and generate high quality data, though this cannot be guaranteed. It is possible to see lower quality, lower data output (lower cluster density or percent occupancy), or run failure.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3444), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003444%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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