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Sequencing run parameters and setup for Illumina Protein Prep (IPP) libraries

What are the supported instruments and flow cells?

  • NovaSeq 6000, S4 (35 cycles). Standard workflow only (no XP/no lane splitting)

  • NovaSeq X Series, 10B (100 cycles).

  • NovaSeq X Series, 25B (100 cycles). The NovaSeq X must be on v1.3+ of the control software. (Custom sequencing recipes are not compatible with earlier versions of the control software.)

What are the recommended read lengths?

  • Read1 = 15

  • Index1 = 10

  • Index2 = 10

  • Read2 = 0

  • Note: the custom recipe must be used for this library type when sequencing.

How much PhiX is recommended? Is it acceptable to add 1-2% for sequencing metrics?

PhiX spike-in is NOT recommended. The software will not report the error rate metric for Read1 lengths under 25 bp, therefore adding PhiX will not provide additional sequencing metrics, and will only take away reads from samples.

What is the recommended final loading concentration per flow cell?

  • Loading concentration guidance can be found in the denature and dilute guides, as well as in the Illumina Protein Prep Product Documentation. Final loading concentration general recommendations are as follows, though loading concentrations may need to be optimized in each lab:

    • NovaSeq 6000, S4: 200 pM (1 nM starting concentration)

    • NovaSeq X Series, 10B: 65 pM

    • NovaSeq X Series, 25B: 85 pM

How many samples per run/lane are supported?

  • NovaSeq 6000: 192 samples per S4 flow cell, standard workflow only

  • NovaSeq X Series 10B: 192 samples over 8 lanes, OR 2x 96-plex pools over 4 lanes each

  • NovaSeq X Series 25B: 384 samples over 8 lanes, OR 4x 96-plex pools over 2 lanes each

  • Note: Illumina Protein Prep libraries must not be mixed with other library types due to their short read lengths and custom sequencing recipe requirements.

Can a one plate of plasma samples and one plate of serum samples be run together in one sequencing run?

Yes. It is important to make sure the correct manifest files/calibrator IDs are used for the sample type but it is possible to combine different sample types in one sequencing run.

For any feedback or questions regarding this article (Illumina Knowledge Article #9802), contact Illumina Technical Support [email protected].

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