Aim: HLA typing for use in transplantation has focused on 11 loci: A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1. AlloSeq Tx17™ is a hybrid capture HLA typing assay that utilizes probes to sequence all these loci with additional probes to include HLA-E, HLA-F, HLA-G, HLA-H, MICA and MICB. In the current Tx17 assay, there are gaps in class II intronic coverage which can result in occasional ambiguities. Alloseq Tx11 was developed with clinical transplantation in mind to increase the sequencing coverage of class II loci, without decreasing the sample throughput for clinical sequencing runs.
Method: Probe sets for the hybrid capture assay were redesigned to include new probes covering intron 2 of DRB1, DRB3/4/5, DQA1, DPA1, and probes covering both intron 1 and 2 for DQB1 and DPB1. These new probes increase the sequencing coverage to full gene for all loci except for intron 1 for DRB1 and DRB3/4/5. Probes for the additional loci outside of the classical 11 were removed from the probe pool. 178 unique samples were tested using the existing AlloSeq Tx workflow, but with the new Tx11 probes used for hybridization. The samples were comprised mostly of DNA sourced from peripheral blood; however, buccal swabs were tested as well. DNA concentrations were determined by Nanodrop spectrophotometry and Qubit fluorescence. Six sequencing runs were completed using a nano flow cell (1), micro flow cells (2), and standard flow cells (3). HLA typing results determined using Tx11 probes were compared to previously known typing results, either from previous Tx17™ runs or external data sources such as IHW or UCLA.
Results: Initial DNA concentrations ranged from 4.25 ng/ul to 58.4 ng/ul; total input of DNA into library prep ranged from 42.5 ng to 584 ng. Results exhibited 100% concordance for the 11 loci that were tested as compared to the known typing. Additionally, some Class II ambiguities traditionally seen with AlloSeq Tx17™ were resolved when using the Tx11 probes. This was due to the ability to phase across introns for Class II that was not previously achievable.
Conclusion: The new intronic probes included in the probe pool for Tx11 successfully increase the intronic coverage for classical class II HLA alleles. This increase in coverage reduces ambiguities and can lead to more confident allele calls in the clinical lab.
