# Best practices for low diversity sequencing on the NextSeq 500/550 and MiniSeq systems

Accurate and robust sequencing of [low diversity or unbalanced composition libraries](https://support.illumina.com/bulletins/2016/07/what-is-nucleotide-diversity-and-why-is-it-important.html) on the NextSeq 500/550 and MiniSeq requires well-designed experiments and informatics pipelines. A common example of a low diversity library is an amplicon-based library preparation method, such as 16S metagenomics. These libraries tend to have DNA sequences that start at the same location - the probe binding site - and are mostly identical. A single represented locus causes a biased base composition that can change drastically from cycle to cycle.

The NextSeq 500/550 and MiniSeq systems use [2-channel sequencing chemistry](https://www.illumina.com/science/technology/next-generation-sequencing/sequencing-technology/2-channel-sbs.html). Therefore, it is important to have all 4 DNA bases represented in every cycle, in order for the software to correctly identify DNA clusters and perform accurate base calling. To meet this requirement using a low diversity library, we recommend experimental designs that provide cycle-to-cycle diversity using the following methods:

* Use indexing to add multiple, indexed samples from various applications to the run.
  * Indexed samples from applications that are more diverse, such as human amplicon sequencing, enrichment, or whole-genome sequencing, can be used to balance diversity.
  * For best results, sequence multiple samples on the same flow cell using single- or dual-indexing.
* Spike-in [PhiX control V3 Library](https://support.illumina.com/bulletins/2017/02/what-is-the-phix-control-v3-library-and-what-is-its-function-in-.html) to the run.
  * A good starting point is to use 50% [PhiX](https://www.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents/phix-control-v3.html) and then titrate the amount down, based on the quality of the primary and secondary analysis results. The presence of the spiked-in sample provides the necessary cycle-to-cycle base diversity.
* Aim to [target a cluster density 30-40%](https://support.illumina.com/bulletins/2017/02/how-much-phix-spike-in-is-recommended-when-sequencing-low-divers.html) beneath the recommended cluster density range for balanced libraries (such as PhiX) on NextSeq and MiniSeq systems.
  * MiniSeq reagents accommodate an optimal raw cluster density of 170-220 K/mm2 for balanced libraries
  * NextSeq 500/550 reagents accommodate an optimal raw cluster density of 170-220 K/mm2 for balanced libraries

The design recommendations in this bulletin enable the NextSeq 500/550 and MiniSeq systems to sequence low diversity libraries.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2882), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002882%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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