OR04: A MULTI-CENTER STUDY USING NEXT-GENERATION SEQUENCING (NGS) FOR HLA GENOTYPING

Aim NGS is well-suited for HLA typing as it can deliver highly accurate and unambiguous results. A simplified protocol has been developed for use with the Illumina MiSeq and has been subjected to an inter- Methods: Six laboratories, each with varying levels of NGS experience, participated in this double-blinded study. The same 16 samples were typed at HLA-A, B, C, DRB1, and DQB1 by each lab. The protocol consisted of LR-PCR, library prep, and paired-end 250 bp sequencing. Two indexing strategies were employed: (1) Locus-specific indexing by which each locus was tagged uniquely and (2) sample-specific indexing whereby all 5 loci for a sample were pooled prior to library prep. Sequence analysis was performed with Target HLA (Omixon) and NGSengine (GenDx) to assess complementarity of two analysis algorithms. Results Six sequencing runs yielded an average output of 7.8 Gb per run. The average number of sequence reads per library was 387,813. However, analysis was limited to 40K reads for the locus-indexed libraries and 200K reads for the sample-indexed libraries (amplicon pools) resulting in an average coverage of 1444. A sufficient number of reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy is shown in Table 1. Reproducibility between labs was 99.7%. The only cause of discordance between labs was cross-contamination of a single amplicon. Conclusion The protocol developed for the MiSeq is reproducible, highly accurate, and its simplicity makes it amenable to clinical testing. The ability of the software to decipher reads from multiple loci in a single library demonstrates the potential for using an amplicon pooling approach that further simplifies sample prep and reduces expense. As demonstrated in this study the protocol is suitable for moderate test volume. However, since 100 samples can be typed in a single run using this approach. A. Berces: Employee; Company/Organization; Omixon, Inc. T. Hague: Employee; Company/Organization; Omixon, Inc. G. Horvath: Consultant; Company/Organization; Omixon, Inc. R. Kooter: Employee; Company/Organization; GenDx . W. Mulder: Stock Shareholder; Company/Organization; GenDx . M. Penning: Employee; Company/Organization; GenDx. K. Rigo: Employee; Company/Organization; Omixon, Inc. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx.