Accurate and robust sequencing of low diversity or unbalanced composition 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:
The design recommendations in this bulletin enable the NextSeq 500/550 and MiniSeq systems to sequence low diversity libraries.