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Troubleshooting low cluster density on the MiSeq
There are a number of possible causes for low cluster density, but the effect is the same. Each cluster gives a read/read pair; if there are fewer clusters than expected, there will be less data generated for analysis.
For MiSeq v2 reagent chemistry, the supported raw cluster density for well-balanced libraries is 1000-1200K/mm2. For MiSeq v3 reagent chemistry, the supported raw cluster density for well-balanced libraries is 1200-1400K/mm2. For low diversity libraries, Illumina recommends targeting a cluster density 30-40% beneath the optimal range for the chemistry version and platform used. The optimal amount of reduction required must be empirically determined. Very low cluster density (less than 500K/mm2) can result in problems registering images and lead to focus errors during the run.
Cluster density is dependent on the amount of library template that is loaded and the clustering efficiency of the library type.
Common causes of low cluster density include:
- Inaccurate quantification of the library.
- Unsuccessful/incomplete library denaturation with sodium hydroxide (NaOH).
- Reagent cartridge storage or handling.
- A variety of instrument issues.
Note: low cluster density is distinct from a clustering failure where no clusters are visible*.*
Persistent clustering failures are usually caused by one of three things--instrument issues, user error, or reagent problems.
Instrument issues typically occur in the fluidics path for the template loading position or reagents used in the early clustering steps. If the cluster density is low but the quality is high, the clustering chemistry and SBS chemistry steps were likely successful.
Additional causes of low cluster density include inaccurate library quantification, inappropriate loading concentration for the library type, use of acidified NaOH for denaturing the library, or NaOH that is too concentrated, which inhibits flow cell binding.
- Library quantification
Library quantification is critical to optimizing data output and quality. Accurate quantification of libraries is essential to optimize the loading concentration.* qPCR is the most sensitive and accurate library quantification method for most library types (not all). qPCR requires additional equipment and reagents. PhiX is not recommended for use as the standard. qPCR has the advantage of only quantifying library molecules with adapters on both ends that can cluster.
- Fluorescent intercalating dye quantification, such as the Qubit assay, is accurate if performed correctly. Fresh standards must be made daily, and pipettes must be calibrated regularly. Dye quantification will not measure single-stranded DNA but will include double-stranded DNA molecules that have incomplete adapters and will not form clusters.
- Illumina does not recommend using trace instruments for the quantification of most library types.
- Spectrophotometric quantification does not give accurate enough concentrations to be useful for the quantification of libraries.
- Sequencing libraries must be stored in the freezer in low-bind tubes or plates.
- Denaturation step
It is important to use NaOH at the right concentration when denaturing libraries. If the NaOH is not at the correct concentration, it will have a lower pH and not denature the library into single strands. Double-stranded libraries will not anneal to the flow cell and will not cluster.
- NaOH must be diluted to 0.2 N, giving a pH of 12.5. Prepare the NaOH dilution fresh regularly, as it will acidify quickly at this concentration.
- NaOH stock solutions can be pH tested with a pH meter or pH strips for alkaline solutions. If the pH of the NaOH stock solution is below 12.5, the diluted 0.2N NaOH will not be able to denature libraries efficiently enough to maximize cluster density.
- It is also possible to have too much NaOH in a denaturation reaction. This can occur when loading high concentrations of libraries that are not diluted sufficiently. Higher concentrations of NaOH in the library inhibit library hybridization to the flow cell and decrease cluster density.
- Consumable expiration dates
Check the expiration date of the reagent cartridge and flow cell. The use of expired reagents or flow cells can result in a variety of issues, including low or no cluster density or intensity and poor data quality. Verify reagent thawing and handling prior to sequencing. Reagents that are expired or that have been thawed outside of Illumina validated guidelines may not perform as expected, and performance is not guaranteed.
- Check the MiSeq chiller temperature
Check the temperature of the MiSeq reagent chiller (on the Sequence screen of the MiSeq Control Software). If the chiller has been opened in the last hour, wait an hour before checking the temperature to ensure that the chiller has had a chance to come to temperature (2°C- 11°C).
- System check
A volume test checks for obstructions in the fluidic lines of the MiSeq. This test requires a used flow cell, a wash tray, and a wash bottle filled with laboratory-grade water. The thermal ramp test checks the thermal station under the flow cell for proper function. If this thermal station is not reaching the correct temperature, the chemistry on the flow cell will not be carried out properly. Additional Resources: * Cluster Optimization Overview Guide
For further information, search for Knowledge Base articles:* How to achieve more consistent cluster density on Illumina Sequencing Platforms
- How to thaw and store sequencing reagents for optimal performance
- Cluster density guidelines for Illumina Sequencing Platforms using non-patterned flow cells