Nucleotide Diversity

Illumina bases cluster density recommendations on diverse libraries. Diversity describes the proportion of each nucleotide (A, T, C, and G) at each position in a library. Low-diversity libraries compromise software performance and data accuracy.

A balanced or diverse library has equal proportions of A, C, G, and T. Low-diversity libraries, such as PCR amplicon, metagenomic, and ChIP, have an uneven proportion of nucleotides across the flow cell from one cycle to the next. Unbalanced libraries, such as bisulfite-converted libraries, have one base at a much lower percentage than the others.

Cluster Images and Data by Cycle

Diverse libraries—Equal proportions of A, C, T, and G with even, horizontal curves centered on 25%.
Low-diversity libraries—Uneven proportions of A, C, T, and G with large intensity spikes at each cycle.
Unbalanced libraries—A low percentage of A and a high percentage of C.

Sequencing Low- and High-Diversity Libraries

When sequencing low-diversity libraries, use the following strategies to increase nucleotide diversity and provide a balanced signal:

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Reduce the loading concentration of the library. Empirically determine the reduction amount.

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Spike in PhiX or other high-diversity library. Empirically determine the spike-in amount, using the following percentages as a general guideline. Start with a higher spike-in percentage and reduce based on run performance.

System	PhiX Spike-In for Low Diversity (%)
HiSeq 2500	≥ 10
HiSeq 3000 and HiSeq 4000	5–20
HiSeq X	5–20
iSeq 100	≥ 5
MiniSeq	10–50
MiSeq	≥ 5
NextSeq 500 and NextSeq 550	10–50
NovaSeq 6000	~5

Although providing a balanced signal is not a concern for high-diversity libraries, Illumina recommends a 1–2% PhiX spike-in as a positive control for sequencing.