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
- 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,
500/550, and MiniSeq.