Best practices to improve data yield when using patterned flow cells on the HiSeq 3000/4000/X

Use the following best practices to ensure successful clustering on patterned flow cells. Inefficient clustering on patterned flow cells can produce focusing errors, low intensities in Read 1, an unusual percent base profile, and poor pass filter rates on the HiSeq system.* Before starting the library denaturation and ExAmp reactions, complete the cBot wash, ensure that the cBot plate is fully thawed, and load the flow cell and manifold onto the cBot.

• After thawing, invert each tube of EPX1, EPX2, and EPX3 five times and then briefly centrifuge. Do not vortex EPX reagents or master mix.

• Aspirate and dispense the viscous EPX reagents slowly to ensure accurate pipetting.

• Add EPX reagents in the order listed (EPX1, then EPX2, and then EPX3). Attach the EPX master mix tube to a lab rotator and rotate for 5 minutes at room temperature to ensure proper mixing.

• Immediately add EPX mix to denatured template and then gently tap the tube strip on a hard surface to remove any air bubbles.

• Promptly start the cBot run.

For detailed information about cluster generation and sequencing using patterned flow cells, see these additional resources:

cBot System Guide HiSeq X System Guide HiSeq 3000 System Guide HiSeq 4000 System Guide

For any feedback or questions regarding this article (Illumina Knowledge Article #1515), contact Illumina Technical Support [e-mail].