NovaSeq X Plus

Available Kits
Each kit is defined by the number of reads (single-end or clusters) and read length (determined by cycle count).
e.g. 10B, 300 cycle flowcell will deliver about 10 billion single-end reads sequenced with the following read structure: 150-10-10-150

Sample Volume Requirements

• 1.5B Flow Cell (2 lanes): 100 µl per lane (min 50 µl)
• 10B Flow Cell (8 lanes): 100 µl per lane (min 50 µl)
• 25B Flow Cell (8 lanes): 140 µl per lane (min 70 µl)

Library Concentration
Submit libraries at 2 nM. CTG will dilute the pool to the final loading concentration in RSB (provided by Illumina), which must be specified in your iLab submission. Loading concentration refers to the final concentration of library loaded onto an instrument for sequencing. See Illumina protocol for details.

Note: Loading concentration must be optimized for your library type. It affects run quality, cluster density, Q30 scores, and total data output. Poor optimization can reduce data quality or cause run failure.

Library Quality & Quantification

• Clean up libraries thoroughly to remove adapter/primer dimers and partial constructs.
• Use a Bioanalyzer, TapeStation or Fragment Analyzer to check fragment size and purity.
• Quantify libraries using qPCR for the most accurate measurement of usable fragments.
• Qubit can be used, but may include unwanted DNA (e.g., adapter dimers).

CTG can offer advice but is not responsible for quantification or concentration optimization.

Sample Delivery

• Sequencing pools delivered by Friday will be scheduled for sequencing the following week. The exact sequencing date is determined each Monday, and you will be notified of the scheduled date on the same day.
• Feel free to deliver your sequencing pool to our lab whenever it suits you, the lab is open all the time. Leave the pool in the black box inside the freezer which is located in lab D1426a and is marked for delivery of SeqOnly pools.
• Label the tubes clearly with the CTG project ID as generated in iLab.
• Upload the sample sheet in the corresponding project in iLab at the same time!

Sample Sheet Submission

A sample sheet (.csv) must be submitted with your library pool. This tells us how to demultiplex your data into FASTQ files. Use the Illumina template provided in iLab. Incorrect formatting may delay your run. Use letters (a-z) and numbers (0-9) only. No spaces, underscores, periods or hyphens.

Include the following fields:

• Sample_ID: Sample name
• Index: i7 sequence (forward)
• Index2: i5 sequence (forward)
• Sample_Project: iLab project number

Lane Assignments:

• Different pools per lane? Assign samples per lane:
  • 1: Sample_A, Sample_B, Sample_C
  • 2: Sample_D, Sample_E, Sample_F
• Same pool across lanes? List each sample with all lanes:
  • 1,2,3,4,5,6,7,8: Sample_A
  • 1,2,3,4,5,6,7,8: Sample_B

Index Sequences Refer to your index kit documentation for correct sequences.

More Info:

• NovaSeq X / X Plus Flow Cell Specs