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
- 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
- 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,
- 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
- 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
- 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.
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,
500/550, and MiniSeq.