To determine the appropriate level of multiplexing, you need to know the output needed for a sample, and how the sequencing run is expected to achieve that. Considering the following:
• The sensitivity (depth of coverage) required per sample.
• The total size and/or the number of regions of interest.
• The expected number of reads and/or bases from the sequencing output, like number of reads per lane or per flow cell.
• Any additional filtering which is applied to the output, for example, more stringent quality filtering, removal of duplicates.
When you have determined the appropriate level of multiplexing, you can start designing the pooling scheme.