Abstract
After a year of implementing HLA typing by NGS, we present our most relevant achievements and challenges.
Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLA-DQ, and Exon 2 thru 3’UTR of HLA-DP) amplified with NXType™ Class I and Class II primer sets. Automated template preparation and chip loading were performed in the Ion Chef™ system and run on Ion S5™. Data was analyzed with TypeStream™ v1.1.0.11 software.
Having typed over 1000 samples by NGS, with 50% from bone marrow programs and 50% from solid organ programs, our workflow has changed significantly: Threefold increase in capacity with simultaneous decrease in turn-around-time (TAT, on average 10days) for high resolution typing. Our current capacity is up to 96 HLA typings in 3–4days vs. minimum of 16days for the same volume with SBT by Sanger. Resolution of most ambiguities (98.7%), minimizing the need for reflex testing.Repetitions due to technical failures and ambiguities decreased from 4.3% with SBT-Sanger to 0.3% with NGS. In spite of positive workflow changes, there remain challenges: Automation of library preparation has not been implemented.No merging of library preparation reagents into a single kit.Smaller runs increase the number of reads, leading to artifacts and high background generated by the software, interfering with analysis and limiting flexibility for STAT sample processing.For solid organ typings, generating high resolution results while reporting Serological Equivalents for UNOS, poses logistical difficulties.TypeStreamTM software not interfaced with Fusion, requiring continuous need to run SSOP for DSA analysis.Unresolved ambiguities (1.3%), due to incomplete exon coverage and phase ambiguities. Resolution may be obtained by increasing gene coverage, length of reads and with additional testing methods.
We have confirmed that NGS is both a suitable and faster method to obtain high resolution HLA typing. We have seen a significant decrease in TAT along with a significant cost savings due to fewer repetitions, fewer reagents, and most significantly, reduced time from our most valuable asset, our technologists.
