Accurate and robust sequencing of low diversity libraries 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. 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 a whole-genome sample (eg, PhiX) to the run.
  • A good starting point is to use 50% PhiX 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 load within the recommended cluster density range for the NextSeq 500/550 and MiniSeq systems per the Cluster density guidelines for Illumina sequencing platforms support bulletin.
  • MiniSeq reagents accommodate an optimal raw cluster density of 170-220 K/mm2.
  • NextSeq 500/550 reagents accommodate an optimal raw cluster density of 170-220 K/mm2.

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