Clustering densities for standard and non-standard library preparation applications

illumina_cluster_generation

Illumina sequencing follows three very simple steps:

  1. Libraries are prepared from DNA or RNA samples
  2. Single molecular DNA templates are bridge amplified to form clonal clusters inside a flow cell
  3. Clusters are sequenced by massive parallel synthesis

Template molecules are immobilized on a flow cell surface and amplified by isothermal bridge amplification to create  individual dense clonal clusters containing ~2,000 molecules each (see figure above).

The exact density of these clusters can influence:

  1. Run quality
  2. Reads passing filter
  3. Q30 scores
  4. Total number of reads

This makes proper loading of an Illumina flow cell crucial to the success of a sequencing run.

In a recent guide, we review recommended loading concentrations and cluster densities for each Illumina instrument. See a summary in Table 1. below:

Illumina flow cell loading recommendations by instrument

While this table includes recommendations for standard library applications where libraries are sufficiently diverse, researchers shouldn’t follow these recommendation for libraries that have poor diversity. Sequence diversity refers to the balance of nucleotides (A, T, G, C) at each position of a template library. Applications where you should load a library at a concentration below Illumina’s standard recommendations include:

  1. Any amplicon based library where primers are included in the read insert
  2. GBS or RAD-seq libraries that start with a similar restriction site
  3. 16S or 18S libraries that start with the same primer or variable domain sequence
  4. MeDIP or other low diversity targeting approach

If you’re working with a non-standard library preparation application or one where libraries have poor sequence diversity, submit a request here: genohub.com/ngs and a scientist will recommend flow cell loading concentrations.

 

 

2 thoughts on “Clustering densities for standard and non-standard library preparation applications

  1. “Hundreds of template molecules are immobilized on a flow cell surface and copied using hybridized by 3’ extension. These immobilized template copies are then amplified by isothermal bridge amplification to create millions of dense clonal clusters (diameter of 1 micron or less) containing ~2,000 molecules.”
    This suggests that few template molecules are amplified to create millions of clusters. This is not the case (at least not the way it is supposed to work). 1 template is supposed to create 1 cluster. Just the number of molecules per cluster is supposed to increase during bridge amplification.

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