Automated size selection of NGS libraries for HLA diagnostics

HLA genotyping is an important procedure in the preparation for stem cell transplants. It is essential for determining the genetic compatibility of the human leukocyte antigens (HLA) between donor and recipient. A high degree of compatibility significantly reduces the risk of rejection and enhances transplantation success. Next- generation sequencing (NGS) offers high-resolution, precise identification of HLA alleles, making it an invaluable method in stem cell transplantation. Accurate HLA genotyping is paramount for successful outcomes in this procedure.
Automating the sample preparation for HLA genotyping provides numerous advantages, such as increased efficiency, reduced error rates, and minimized manual handling time. During the library preparation stage for NGS, the cleanup step is crucial. This step involves replacing the buffer containing DNA fragments and removing remaining enzymes and components from previous preparation stages, like primers and adapters. Various methods have been developed for DNA fragment cleanup, with the use of magnetic beads being the most straightforward.
Magnetic beads with their specific coating and optimized buffer conditions allow for the binding of DNA molecules. The size of the bead-bound DNA fragments largely depends on the concentration of the buffer components the beads are stored in. By adjusting the bead-to-sample ratios, DNA fragments within a user-defined size range can be selectively isolated. In this context, a double-sided size selection was performed using the liquid handling robot CyBio FeliX to extract fragments between 500 bp and 800 bp for subsequent sequencing.
During library preparation, DNA concentration measurements were performed before and after size selection as well as fragment size assessments after sequencing, using the NGSengine software (GenDx) to evaluate the efficacy of the size selection steps. The reduction in DNA concentration following the 0.7x bead clean up and size selection procedures indicates successful removal of unwanted DNA fragments and contaminants. For the 0.7x bead cleanup, the average DNA concentration decreased significantly (t-test, p > 0.05), with differences ranging from -4.4 to -34.6 ng/μL across all samples, demonstrating effective purification via the automated workflow. Similarly, size selection also resulted in a consistent reduction in DNA concentration, ranging from -2.4 to -15.4 ng/μL, targeting the isolation of DNA fragments within the desired size range (600-800 bp). These results confirm that the cleanup and size selection processes efficiently enrich the samples for high-quality and appropriately sized DNA fragments, essential for optimal sequencing performance.
The fragment sizes in the automated workflow were on average significantly larger (t-test, p < 0.05) ranging between 250 bp to over 350 bp compared to the manual process ranging between 210 bp to 320 bp, aligning more closely with the goal of size selection. Automating processes therefore ensure reliable and efficient selection of correct size fragments optimal for sequencing procedure downstream. The advantage of the automated process over the manual process on CyBio FeliX is that all samples can be processed simultaneously without any time delay. This prevents the excessive binding of smaller fragments to the beads. Accurate adherence to incubation times ensures correct size selection of the fragments.
Optimal conditions for sequencing are ensured by correct size selection, providing ideal sequencing conditions. The efficiency of the automated system is beneficial, as it can perform multiple cleanups sequentially, speeding up the process compared to manual methods. Improved accuracy is another advantage, as manual methods are prone to errors, whereas automation enhances reproducibility. Lastly, the ergonomics of automation reduce the physical strain associated with manual handling, making the process more user-friendly.
The automated workflow, with its ability to consistently produce larger DNA fragments and handle multiple samples efficiently, not only aligns with sequencing requirements but also offers significant benefits in terms of throughput, reproducibility, and error reduction. This ensures optimal conditions for sequencing and enhances overall process efficiency.
To summarize, the use of the CyBio FeliX allows 96 samples to be processed simultaneously, in approximately 40 minutes, with the flexibility to process multiple runs during the day. The compact device reduces the bench space needed for an automated library preparation process for optimal sequencing on an Illumina® NovaSeq. Using the CyBio FeliX for these procedures makes them 4x faster compared to the manual workflow. In addition, automation with CyBio FeliX increases walk-away time significantly.