P114 Validation of high resolution HLA typing using NXTypeTM Ion-Torrent next generation sequencing. The University of Miami experience

Aim HLA typing using next generation sequencing (NGS) takes the Sanger based sequencing HLA typing to a higher dimension. The technology permits multiplexing of several target regions and various samples in a single run. The advantages of this technology include high throughput, improved DNA sequencing accuracy and faster data analysis. Our aim was validate the NXTypeTM Ion-Torrent NGS HLA typing procedure to implement it in our clinical laboratory. Methods 214 samples were analyzed with NXType™ Ion-Torrent NGS and compared to high resolution typing obtained by Sanger sequencing. HLA 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) were amplified with NXType™ Class I and Class II primers set, respectively. Amplicons were fragmented with Ion Shear™ Plus Reagents Kit to sizes from 350 to 950bp. Libraries were enriched in OneTouch ES and run in an Ion PGM™. Data was analyzed with HLATypeStream (v1.0.0.86) software. Results We obtained good coverage of the target regions when multiplexing 24 samples per run using a 318™ v2 chip. Of the 1070 alleles analyzed (214 samples), 143 were homozygous and 927 heterozygous. The concordance between Sanger sequencing results and NGS was 99.2%, 99.6%, 99.8%, 100% and 99.8% for loci A, B, C, DRB1 and DQB1, respectively. The number of different alleles identified for loci A, B, C, DRB1 and DQB1 were 40, 69, 36, 42, and 18, respectively. Our results revealed that 269 NMDP codes reported by Sanger sequencing were resolved by NGS, except 3 codes in DRB1 because of mismatches present in exons 1 or 4, which are not targeted by NXType™ Class II primers. We found two new alleles in exons (A ∗ 24, exon 1 and C ∗ 01, exon 2) and variations in intronic regions of 46 alleles. All null alleles A ∗ 68:11N (10), B ∗ 15:01N (11) and C ∗ 04:09N/B ∗ 44:03 (3) seen by Sanger were resolved by NGS. Conclusions The NGS is a reliable and accurate method to determine HLA typing of a considerable number of samples relatively fast, solving most allele ambiguities (98.7%) without the need of alternative tests. We confirmed that NXTypeTM Ion-Torrent NGS is a suitable and quick method for routine use in clinical laboratories to obtain complete ultra-high-resolution HLA typing that ultimately will improve on current HLA matching for transplant.