P082 : IMPLEMENTATION OF NEXT GENERATION SEQUENCING (NGS) TECHNOLOGY FOR HLA TESTING: KEY LESSONS LEARNED FROM A MULTI-CENTER ALPHA STUDY

Abstract

Background NGS technologies have significant potential to generate high resolution HLA typing in a high throughput fashion. However, implementation in clinical immunogenetics laboratories involves extensive validation of a multitude of interdependent procedures, instruments, reagents, and software. To address these challenges we participated in March of 2014 in a multicenter Alpha study lead by the Immunogenetics Laboratory of a large academic institute that has developed an NGS-based protocol for HLA typing on the Illumina MiSeq. Methods We participated in the Alpha study with 2 main objectives: (1) validate the in-house developed NGS genotyping protocol for the HLA-A, B, C, DRB1 and DQB1 loci; (2) identify key critical steps in the protocol that we had problems with in our own lab prior NGS implementation. This abstract focuses only on key technical lessons learned from the study. The Alpha study results are independently submitted in another abstract. Results Participation in the Alpha study identified some critical steps in NGS workflow: (1) the quantitation of genomic DNA needs to be assessed by using methods that directly measure double stranded DNA concentration during primary sample preparation prior to target enrichment, (2) the library preparation reagents need to ensure adequate and uniform coverage throughout the length of the HLA targeted genomic region, (3) use appropriate methods for fragment size selection including magnetic beads and/or specialized electrophoretic instrumentation after library preparation, (4) quantitation and normalization of DNA of each amplicon to a standard DNA amount prior to amplicon pooling, (5) use two independent genotyping algorithms for data analysis to avoid systematic errors of genotype assignments caused by individual analysis software. Specific details about these lessons learned and more technical tips will be provided in this presentation. Conclusion In spite of the technical complexity of NGS workflow, identifying key critical steps in the process and identifying troubleshooting strategies are critical in implementing NGS in the clinical laboratories. Inter-laboratory validation studies and seeking the guidance from laboratories who developed NGS expertise is invaluable for expediting the learning curve for the implementation of this technology. M. Askar: Employee; Company/Organization; Cleveland Clinic.