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

See below for an overview of the denature and dilution process for the NextSeq 500/550.

Loading Volume and Concentration

Denature and dilute libraries to a final loading volume of 1.3 ml at a 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.

• In practice, loading concentration can vary depending on library preparation and quantification methods. Library loading concentrations must be optimized within the lab.

Protocol Variations

Follow the appropriate denature and dilute protocol depending on the procedure used during library prep.

• Standard normalization (Protocol A): Libraries are normalized using standard library quantification and quality control procedures recommended in the library prep documentation.

• Bead-based normalization (Protocol B): Libraries are normalized using a bead-based procedure described in the library prep documentation for methods that support bead-based normalization.

• AmpliSeq for Illumina normalization (Protocol C): For all AmpliSeq for Illumina panel libraries, follow Protocol C.

Best Practices

• Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. 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.

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

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

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