Low diversity sequencing on the MiniSeq

Low diversity sequencing requires well-designed experiments to succeed. Common examples of low-diversity libraries include amplicon-based library preparation methods such as 16S metagenomics. These libraries tend to have DNA sequences that start at the same location (the primer binding sites) and are mostly identical. A single represented locus causes a biased base composition that can change drastically from cycle to cycle.

As the MiniSeq system uses 2-Channel Sequencing Chemistry, it is critical to have all DNA bases represented in every cycle so that the software may correctly identify clusters and perform accurate base calling. To meet these requirements, Illumina recommends using the following methods when sequencing low diversity libraries.

• Illumina recommends adding 10-50% PhiX to increase diversity.

  • When sequencing a new library type, Illumina recommends starting at 50% PhiX spike-in, then titrate the amount down, based on the quality of the primary and secondary analysis results.

• Illumina recommends reducing the target cluster density by 30-40% for low-diversity libraries. For a MiniSeq, this means targeting 102-154 K/mm2 (reduced from the normal guidelines of 170-220 K/mm2).

For any feedback or questions regarding this article (Illumina Knowledge Article #2036), contact Illumina Technical Support [e-mail].