Pool and Denature Libraries

Use this protocol to denature and dilute Illumina Cell-Free DNA Prep with Enrichment libraries prepared using the bead-based normalization option. Libraries prepared using the bead-based normalization option are ready for sample pooling.

The NovaSeq X Plus 10B Flow Cell has eight lanes and the NovaSeq X Plus 1.5B Flow Cell has two lanes. Use this protocol for sequencing a single pool across both lanes of the 1.5B Flow Cell or a single lane of the 10B Flow Cell.

Preparation

1.

If the normalized library (NL) plate was stored, thaw to room temperature.

2.

Centrifuge the plate at 280 × g for 10 seconds.

3.

Preheat the heat block to 96°C.

4.

Prepare an ice bucket.

Pool Normalized Libraries

1.

Calculate library plexity for sequencing. Select the appropriate plexity calculation factor.

2.

Divide the plexity calculation factor by the panel size for enrichment in megabases (Mb).

This calculation is a recommended guideline. If each sequenced library does not achieve at least 30,000x on target coverage, reduce the number of libraries in the pool.

3.

Set a pipette to 30 μl, and then gently pipette to mix the libraries in the NL plate 5 times.

Use fresh tips for each library.

4.

Label a 1.5 ml low DNA binding microcentrifuge tube PDL (pooled DNA libraries).

5.

Transfer equal volumes of each normalized DNA library from the NL plate to the PDL tube. The recommended final pool volume is a minimum of 70 µl for a 1.5B flow cell, and a minimum of 25 µl for a 10B flow cell. For example, for a 10-plex library pool on a 1.5B flow cell, combine 7 µl from each bead-based normalized library.

A library generated as a 4-plex enrichment pool is 4x less concentrated than a 1-plex library. Therefore, sequencing pooling by volume should respect that ratio. For example, 20 μl of 4-plex libraries should be pooled with 5 μl of a 1-plex library.

6.

Vortex the PDL tube briefly to mix, and then centrifuge briefly.

Denature Library Pool

1.

Incubate the PDL tube in a heat block at 96°C for 2 minutes.

2.

Invert 2 times to mix.

3.

Centrifuge briefly.

4.

Immediately place on ice for 5 minutes.

5.

Store on ice until use.

Safe Stopping Point

If you are stopping, store denatured libraries at -25°C to -15°C for up to 30 days. To use frozen library pools, thaw the tubes and repeat Denature Library Pool before proceeding to the next step.

Dilute Library Pool

Prepare RSB

1.

Remove RSB from 2°C to 8°C storage, and bring to room temperature.

Dilute Libraries

1.

Label a 1.5 ml low DNA binding microcentrifuge tube DIL1 (Dilution 1).

2.

Vortex the PDL tube briefly to mix, and then centrifuge briefly.

3.

Add the appropriate volume of PDL and RSB to the DIL1 tube as follows.

4.

Vortex the DIL1 tube briefly to mix, and then centrifuge briefly.

5.

Add the appropriate volume of Pre-load buffer as follows.

6.

Vortex briefly to mix, and then centrifuge briefly.

7.

Store libraries on ice until transferred to the library tube strip.

After denaturing and diluting the libraries, you are ready to load the lyo insert and library tube strip into the reagent cartridge in preparation for sequencing. Refer to the sequencing system product documentation for more information.

Revision History - Protocol for Illumina Cell-Free DNA Prep with Enrichment, Bead-Based Normalization

Document	Date	Description of Change
Document # 200055976 v00	June 2024	Initial release.