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Library loading concentrations and considerations for the NovaSeq X/X Plus instruments

Recommended Loading Concentrations
Important Note: The following recommendations are general starting points. The optimal loading concentration depends on the library type and insert size. Titration of each library type is recommended to obtain optimal seeding concentration to yield the best clusters passing filter percentage (%PF). Optimize concentrations over subsequent runs to identify a loading concentration that consistently yields data that meets run specifications.
Dilute and Denature Protocol
Prepare Sodium Hydroxide (NaOH)
  1. 1.
    Combine the following volumes in a microcentrifuge tube. These volumes result in 100 µl 0.2 N NaOH for one flow cell or 200 µl 0.2 N NaOH for two flow cells.
  2. 2.
    Vortex and then centrifuge the tube to mix.
Dilute Libraries and Add Optional PhiX Control
  1. 1.
    Dilute libraries to 2 nM using Resuspension Buffer (RSB).
  2. 2.
    Choose desired final loading concentration and, in a new 1.5 ml microcentrifuge tube, dilute libraries using RSB. The final volume of the libraries should be 34 µl per sample well.
  3. 3.
    [Optional] Spike in 1% to 2% nondenatured PhiX for well balanced libraries.​​​​​​
  • Quantify PhiX stock solution.
  • Dilute 10 nM PhiX to 300 pM using RSB.
  • Add 1 µL of PhiX to 34 µl of nondenatured library that has been diluted to the desired final loading concentration.
Denature Libraries
  1. 1.
    Add 8.5 µl 0.2 N NaOH to the nondenatured library tube and optional PhiX.
  2. 2.
    Cap the tube and vortex briefly.
  3. 3.
    Incubate at room temperature for 5 minutes to denature.
  4. 4.
    Add 127.5 µl Pre-load Buffer to neutralize.
  5. 5.
    Cap the tube and vortex briefly.
  6. 6.
    Centrifuge at 280 x g for up 1 minute.
  7. 7.
    Store libraries on ice until transferred to the provided library tube strip.
On the NovaSeq X/X Plus Instruments, the prepared denatured library volume is 170 µL and the loading volume is 165 µl for each well in the library tube strip, regardless of flow cell type.
Library Pool: 34 µl (35 µL if using optional PhiX) 0.2 N NaOH: 8.5 µl Pre-load Buffer: 127.5 µl Total Volume: 170 µL (171 µL if if using optional PhiX)
For further information about Dilute and Denaturing, visit the Protocol section in the NovaSeq X Product Documentation.
For any feedback or questions regarding this article (Illumina Knowledge Article #7778), contact Illumina Technical Support [email protected].
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