How to achieve more consistent cluster density on Illumina sequencing platforms
Achieving optimal cluster density is critical to high-quality sequencing on MiniSeq, MiSeq, NextSeq 500/550, and HiSeq 2500 Systems. On nonpatterned flow cells, cluster density has a significant impact on run performance, specifically data quality and total data output. While underclustering can maintain high data quality, it results in lower data output. Alternatively, overclustering can lead to run failure, poor run performance, lower Q30 scores, introduction of sequencing artifacts, and lower total data output. This bulletin summarizes the resources and best practices to avoid underclustering and overclustering, and to achieve more consistent cluster densities.
Quality check and accurate quantification of libraries is critical. If not performed, it is the most common cause of inconsistent cluster density. Always follow methods listed in the Library Quantification and Quality Control Quick Reference Guide bulletin to check Illumina libraries.
Follow the latest instrument-specific guidelines to dilute and denature libraries:
Best practices for library denaturation:
The NaOH stock solution must have a pH >12.5 prior to any dilution. Check the pH of the stock solution before diluting. The recommended stock concentration is found in the appropriate instrument system guide or denature and dilution guide.
Always prepare freshly diluted NaOH for denaturing libraries at time of use.
To prevent small-volume pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of freshly diluted NaOH.
For best results, always thaw sequencing reagents before denaturing and diluting libraries. For further instructions, see the system guide for your instrument and the bulletin: How to thaw and store sequencing reagents for optimal performance.
Follow the denature and dilute libraries guide for your instrument. - For HiSeq 2500 and the MiSeq series, it is important that the final solution of NaOH is not more than 1 mM NaOH after diluting with HT1. Greater than 1 mM NaOH inhibits template hybridization efficiency. - When denaturing libraries for NextSeq 500/550 and MiniSeq, use 200 mM Tris-HCl pH 7.0 to make sure that the NaOH is fully hydrolyzed in the final solution. As a result, template hybridization is not affected even when the final NaOH concentration is greater than 1 mM (see NextSeq 500/550 and MiniSeq denature and dilution guides, above).
For highly structured or GC-rich libraries, cluster density consistency improves when the library is heat denatured before loading the pool onto the instrument. This method is optional for other libraries.
After NaOH denaturation of the sequencing library and dilution in HT1 to the final loading concentration, incubate the diluted library at 96°C for 2 minutes using a heat block.
After the heat incubation, invert the tube 1-2 times to mix.
Quickly move the library to an ice water bath for 5 minutes. The quick cooling step helps lock the library in its single stranded form.
Proceed immediately to cluster generation.
Consider the average size of your library. Because the clustering process preferentially amplifies shorter libraries in a mixture of fragments, large libraries tend to cluster less efficiently than smaller libraries. The Nextera Library Validation and Cluster Density Optimization technical note lists guidelines for loading concentrations based on the average size of the library.
Special consideration is needed when sequencing low-diversity libraries to ensure consistent cluster density and good data quality. For low diversity sequencing guidelines, refer to the Technical Note for the appropriate instrument: MiSeq, HiSeq, NextSeq 500/550, and MiniSeq.
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