Automated NGS improves accuracy and efficiency in HLA genotyping for transplants.
Sponsored Content by Analytik Jena USReviewed by Andrea SalazarOct 16 2024 Human leukocyte antigen genotyping is critical when preparing for stem cell transplants. The procedure is crucial to determine the genetic compatibility of the HLAs between the donor and recipient.1 A high level of compatibility considerably minimizes the risk of rejection and improves transplant success.
Magnetic beads allow DNA molecules to bind with their unique coating and optimal buffer conditions. The size of the bead-bound DNA fragments is largely determined by the concentration of the buffer components in which the beads are kept. The automated stages were integrated into the library preparation process of the NGSgo® Library Full Kit for NGS HLA typing. Before library preparation, the NGSGo® Ampx v2 kit was used to perform singleplex PCR on six distinct HLA loci.
Figure 2. Graphical deck layout for Protocol – Double-sided Size Selection. The CyBio FeliX provides 12 deck positions on 3 movable decks. The upper decks B and C equipped with the required accessories are shown on the left, the lower deck A is shown on the right. The 12-column reservoir on position 8 is equipped with 2 mL AMPure XP beads per column. Image Credit: Analytik Jena US
Step 4: Supernatant transfer In a single pipetting step, transfer the supernatant containing unbound DNA fragments into a fresh 96-well PCR plate using a RoboTipTray 96/250 µL. Place the plate on the BioShake using the gripper.Step 6: Setup for bead cleanup From the beads reservoir, 3.9 µL are transferred into each well of the 96 well PCR plate containing the supernatant. The beads are mixed with the DNA containing solution by pipetting five times.
Step 10: Ethanol wash Add 100 µL 80 % ethanol to the beads using a new RoboTipTray 96/250 µL. The beads are incubated with ethanol for 30 seconds while remaining on the well’s wall. Step 13: Final bead separation Place a PCR plate containing resolved beads on the magnet adapter using the gripper and incubate at room temperature for five minutes. Beads should not remain in the solution after incubation.
Results and discussion During library construction, DNA concentration measurements were taken before and after size selection and fragment size evaluations after sequencing using the NGSengine software to analyze the effectiveness of the size selection processes. These findings demonstrate that the cleansing and size selection operations effectively enrich the samples for high-quality, properly sized DNA fragments required for good sequencing performance.
This prevents tiny particles from attaching too tightly to the beads. Accurate adherence to incubation durations guarantees that the fragments are correctly sized. Another advantage is the increased precision, as manual procedures are more prone to errors, while automation enhances reproducibility. Additionally, the ergonomics of automation reduce the physical strain associated with manual handling, making the process more user-friendly.
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