> 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/general/library-preparation-general-reference_material-list/000001705.md).

# Library denaturation and compatibility for the iSeq 100 system

Libraries loaded onto the iSeq 100 system **must** be double stranded DNA, as libraries are automatically denatured into single strands onboard the instrument. Bead-normalized libraries are single-stranded and are not compatible with the iSeq 100.

The [Nextera XT](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-xt-dna.html) library preparation workflow includes an optional bead normalization procedure. When sequencing on the iSeq 100, omit the bead normalization steps and proceed with manual normalization of the libraries.

* For more information on manual normalization, see [Best practices for manually normalizing library concentrations](/library-preparation/general-library-prep/library-preparation-general-library-prep-reference_material-list/000001252.md).

Other library prep workflows listed below also yield single-stranded libraries and are additionally incompatible with iSeq 100 due to output and analysis requirements.

* [TruSight Oncology 500 v2](https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500.html)
* [TruSight Oncology 500 ctDNA v2](https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500-ctdna.html)
* [TruSight Oncology 500 High Throughput](https://www.illumina.com/products/by-type/clinical-research-products/trusight-oncology-500-ht.html)
* [TruSight Tumor 170](https://www.illumina.com/products/by-type/clinical-research-products/trusight-tumor-170.html)

**Exception**

The single exception to the requirement of double-stranded DNA libraries is the [Illumina DNA PCR-Free](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/dna-pcr-free-prep.html?langsel=/us/) library preparation workflow. Although this workflow yield single-stranded DNA, it has been validated on the iSeq 100. iSeq 100 Control Software version 3.0 or higher is required for running this library type, as the libraries require the supplemental sequencing primers VP10 and VP14 for Read 1 and Index Read 2, respectively. These are sold separately and must be loaded as custom primers on board the instrument. See [Custom primer requirements for the Illumina DNA PCR-Free Prep, Tagmentation kit](/library-preparation/general/library-preparation-general-reference_material-list/000003343.md) for additional information.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #1705), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001705%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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