Best practices for low diversity sequencing on the NextSeq 500/550 and MiniSeq systems
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Accurate and robust sequencing of 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 . 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 to the run.
A good starting point is to use 50% 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 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.
For any feedback or questions regarding this article (Illumina Knowledge Article #2882), contact Illumina Technical Support .