P099 : A SINGLE CENTER COMPARISON OF HIGH-RESOLUTION HLA TYPING BY NEXT-GENERATION VERSUS SANGER SEQUENCING

Abstract

Sanger sequencing has been the gold standard for high resolution HLA typing for years. However, typing ambiguities require additional testing to provide allele level resolution. Recently, the development of next-generation sequencing (NGS) technologies for HLA typing has allowed for significantly improved HLA typing. We compared the relative laboratory cost of HLA typing by Sanger sequencing compared and NGS in our moderate volume laboratory. The cost of 30 HLA typings (HLA-A, B, C, DRB1 and DQB1) was compared between Sanger sequencing and NGS. Sanger sequencing was performed with SeCore HLA Typing kits (Life Technologies). Sanger costs included reagent costs for GSSP (SeCore) and/or SSOP (One Lambda) follow up testing for ambiguity resolution. Samples for NGS were amplified using GenDx primers and libraries prepared using Nextera XT kits (Illumina). Sanger samples were run on ABI 3500 while NGS libraries were run on the MiSeq. Analysis was performed using UType software (Sanger) and NGSEngine software (NGS). The reagent cost of Sanger and NGS were based on primers, library preparation and sequencing reagents for each technology. Technologists’ time was an estimation of hands-on time from post-DNA extraction to sequencing data analysis for each method using mean hourly wage. The data was extrapolated to 30 patients, which we anticipate being a typical NGS run. The data doesn’t account for the 7% repeat Sanger sequencing rate we observe for Sanger sequencing run failures. Overall, the cost of HLA typing for 30 patients (5 loci) using NGS was reduced 49% ($22,435) compared to Sanger. Reagent cost for NGS was reduced by $22,073 (49%) compared to Sanger. Technologist time was reduced 60–70% by utilizing NGS instead of Sanger. Sanger sequencing is the gold standard for high-resolution HLA typing however it has limitations. Heterozygous samples often present a problem due to phase ambiguities that require additional testing. The ability of NGS to perform millions of sequencing reads helps correctly phase each nucleotide thereby reducing ambiguities. Moving high-resolution HLA typing from Sanger to NGS will provide substantial savings via multiplexing and reduced reagent cost. Since the cost of NGS instruments and reagents continues to decrease, savings will continue to grow.