Aim: Our lab received approval of the validation of Omixon NanoTYPE HLA typing using Oxford Nanopore Technology (ONT) from ASHI and New York State Department of Health in 2023. During our validation, we identified the main drawback of this method as DQ3 dropouts. Particularly concerning were DQ5/DQ3 or DQ2/DQ3 combinations, with the possibility of DQB1*03 allele dropout being 30.8% or 6.7%, respectively. We started testing patient samples at the beginning of 2024, and closely monitored this issue. This abstract outlines the improvements made by the vendor and presents additional observations.
Method: Our lab routinely uses blood for clinical testing, the Maxwell RSC (Promega) to extract DNA, and the VeritiPro Thermal cycler (ThermoFisher) for DNA amplification. The Omixon NanoTYPE assay involves multiplexed long-range PCR (Nano TYPE 24/11 v2 kit), rapid barcoding library preparation, nanopore sequencing (MinION flow cells), and data-analysis using NanoTYPER software. To date, we have tested 132 patient samples.
Results: Of the 132 samples, 61 had at least one DQ3 allele. Among these, 14 samples had a DQ5/DQ3 combination and another 14 had a DQ2/DQ3 combination. No DQ3 dropouts observed. Seven DQ3 homozygous samples were identified, with five confirmed through SSP high-resolution tests. The remaining two were homozygous for all other class II loci, so no further SSP tests were conducted.
Additional observations: 1. Some rare alleles detected include A*02:742, A*01:06, DRB3*02:24, DRB5*02:21, DQB1*03:42, DPA1*02:12, and two instances of DPA1*03:05:01Q which were verified as correct calls by the vendor. 2. A dropout of B*51:01:02 was observed and confirmed via RT PCR. The vendor identified the issue to a 5’UTR primer mismatch caused by a 3-base insertion in the allele’s binding site. 3. A single DQA1 dropout was observed but successfully amplified up repetition.
Conclusion: Since our initial validation of the NanoTYPE method two years ago, significant improvements in both the amplification kit and software have been made by Omixon, effectively resolving the DQ3 dropout issue and enhancing overall performance.
