Loading Concentration

Loading concentration or final loading concentration is the ultimate concentration of a library loaded onto an instrument for sequencing. After library prep, libraries are diluted to the loading concentration appropriate for the library type, sequencing system, and reagent kit.

• Loading libraries at a concentration that is too high results in overloading or overclustering, which reduces %PF and can cause run failure.
• Loading libraries at a concentration that is too low results in underloading or underclustering, which reduces data output and accuracy.

To determine the optimal loading concentration, adjust the loading concentration in small increments. As a starting point, see the recommended loading concentration in the denature and dilute instructions for your system. Depending on the system, instructions are in the denature and dilute guide or the system guide.

The relationship between cluster density and loading concentration is nonlinear, so small, empirical adjustments are necessary to determine the optimum loading concentration. For example: If a 10 pM loading concentration results in 70% of optimal cluster density, do not assume that loading another 30% achieves optimal cluster density. Achieving optimal cluster density likely requires a smaller increase in loading concentration than 30%.

Sodium hydroxide (NaOH) denatures libraries normalized using standard quantification and quality control procedures. When denaturing libraries with NaOH, use freshly diluted NaOH with a pH > 12.5. Make sure that the final concentration of NaOH in diluted libraries is < 1 mM.

• NaOH that is not freshly diluted can acidify. The resulting decrease in pH impairs denaturation and reduces cluster density.
• Excess NaOH concentration in diluted libraries inhibits cluster formation. Dilute NaOH to the concentration indicated in the denaturation instructions for your system, using Tris-HCl as needed to neutralize the pH.

The iSeq 100 System denatures libraries automatically onboard the instrument, so these considerations do not apply.