How to denature and dilute libraries on the NextSeq 500/550

Loading Volume and Concentration

Denature and dilute libraries to a final loading volume of 1.3 ml at the recommended concentration:

• For the PhiX control library, Illumina recommends a final loading concentration of 1.8 pM for high output kits and 1.5 pM for mid output kits.

• For sequencing libraries, ideal loading concentrations can vary depending on library preparation and quantification methods. If sequencing non-Illumina libraries, users must optimize their loading concentrations internally.

Best Practices

• Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. This step is essential to the denaturation process.

• To prevent pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of freshly diluted NaOH.

• For best results, begin thawing reagents before denaturing and diluting libraries. For instructions, see the NextSeq 500/550 Sequencing System Guide.

For the complete NextSeq 500/550 denature and dilution protocol, see the NextSeq 500/550 Denature and Dilute Libraries Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #2064), contact Illumina Technical Support [e-mail].