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Best practices to avoid low cluster density on the MiSeq
Underclustering (too low of cluster density) can lead to poor run performance, lower Q30 scores, the possible introduction of sequencing artifacts, and lower total data output.
The following sections outline best practices to avoid underclustering on the MiSeq.
Best practices for quantification:
- Accurate quantification and proper quality check of sequencing libraries are key to a successful sequencing run. For further information, search for Knowledge Base article How Do I Achieve Consistent Quantitation?
- Different methods for quantification and quality control are recommended depending on the sequencing library kit being used. For further information, search for Knowledge Base article Library quantification and quality control quick reference guide.
- Consider the average size of the libraries in the run. Because the clustering process preferentially amplifies shorter libraries in a mixture of fragments, large libraries tend to cluster less efficiently than smaller libraries.
Best practices for denaturation and dilution:
- The 2 N NaOH stock solution must have a pH >12.5 prior to any dilution. Check the pH of the stock solution before diluting; the incorrect pH of NaOH can negatively impact the cluster density of a run.
- Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. Use within 12 hours of preparation, if not sooner. This step is essential to the denaturation process.
- To prevent small pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of freshly diluted NaOH.
- If the library pool requires a final concentration > 20 pM, make sure that the NaOH concentration is ≤ 0.05 N in the denaturation solution and ≤ 0.001 N (1 mM) in the final solution diluted with HT1. Higher concentrations inhibit hybridization and decrease cluster density.
- For highly structured or GC-rich libraries, cluster density consistency improves when the library is heat-denatured before loading the sample 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.
Best practices for sequencing low diversity libraries:
- Nucleotide diversity is required for effective template generation on Illumina sequencing platforms and is important for the generation of high-quality data.
- Diversity is especially important during the first 4-7 cycles of the first sequencing read for MiSeq systems. The sequencing software uses images from these early cycles to identify the location of each cluster in a process called template generation.
- A minimum of 5% PhiX is required for sequencing low base diversity libraries on the MiSeq with v3 consumables.
- The PhiX Control Library has a diverse base composition (45% GC and 55% AT) that provides the balanced fluorescent signals that low diversity sample libraries lack during each sequencing cycle. This assists with template registration and improves overall run quality.
- Reduce the library loading concentration to target 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.
See the following for additional information:
- For further information, search for Knowledge Base article(s):
- How much PhiX spike-in is recommended when sequencing low diversity libraries on Illumina platforms?
- How to achieve more consistent cluster density on Illumina sequencing platforms.
- Cluster density guidelines for Illumina sequencing platforms using non-patterned flow cells.
Last modified 2mo ago