Best practices for LNB1 handling in bead based normalization for TruSight Oncology 500 workflows

After sample preparation, accurate normalization of each library to the same concentration for pooling and sequencing is critical. This step makes sure that an equal amount of each library is represented in the sequencing library pool to achieve optimal cluster density, sequencing output, and even data distribution.

The TruSight Oncology 500, TruSight Oncology 500 High-Throughput (HT), and TruSight Oncology 500 ctDNA workflows use a bead-based normalization (BBN) method to normalize library concentrations at the end of sample preparation. When an equal number of library normalization beads (LNB1) is added to each library, and each library has a yield of at least 3 ng/µL, an equal number of library fragments will be captured on the LNB1 beads and eluted for pooling. Therefore, the number of beads added to each library will determine the success of library normalization and subsequent sequencing.

To ensure that an equal number of beads is allocated for each library, complete resuspension of the LNB1 beads is critical. Illumina recommends the following steps when working with the LNB1 reagent:

1. Bring LNB1 to room temperature for at least 30 minutes before use.

2. Pulse-vortex (general lab supply) LNB1 tube for 1 minute at maximum speed.

3. Invert LNB1 tube after vortex to inspect the bottom of tube for bead pellet (Fig.1).

4. If a bead pellet is observed, repeat step 2.

5. Caution: It is critical to completely resuspend the bead pellet at the bottom of the tube. Resuspension is essential to achieve a consistent cluster density.

6. Using a P1000 pipette set to 800 µl, manually pipette up and down 10 times to fully resuspend LNB1 beads to a homogenized solution

7. Combine the LNA1 buffer and LNB1 buffer in a new microcentrifuge tube to create LNA1+LNB1 Master Mix (refer to table in corresponding TruSight Oncology 500, TruSight Oncology 500 High-Throughput (HT), and TruSight Oncology 500 ctDNA reference guide for making LNA1+LNB1 Master Mix):

   1. Vortex LNB1 beads immediately prior to preparation of LNA1+LNB1 Master Mix

   2. If LNB1 is set aside before making LNA1+LNB1 Master Mix, repeat Steps 2­-­5

Figure 1. A full one-minute pulse-vortex is needed for complete resuspension of LNB1.

1. LNB1 pulse-vortexed between 5 seconds and 1 minute are similar in appearance but may vary in degree of bead resuspension.

2. Residual bead pellets are observed at the bottom of the tube when LNB1 is pulse-vortexed for < 1 minute. This phenotype is indicative of incomplete bead resuspension.

For any feedback or questions regarding this article (Illumina Knowledge Article #2721), contact Illumina Technical Support [e-mail].