OR57: GENERATION OF 252 HLA CLASS I GENOMIC SEQUENCES IN A SINGLE SEQUENCING REACTION USING DNA BARCODES AND SINGLE MOLECULE REAL-TIME (SMRT) DNA SEQUENCING TECHNOLOGY

Abstract

Aim To sequence entire HLA class I genes of 45 DNA samples in a single sequencing reaction using DNA-barcode-labelled primers and Single Molecule, Real-Time (SMRT®) DNA sequencing technology. Method Our previous experience of sequencing full-length HLA class I genes using SMRT® technology has shown that it has read length capabilities and accuracy amenable for definitive allele-level resolution HLA typing. Further experiments to determine multiplexing using DNA barcodes have shown that multiplexing up to 20 DNA samples for a single HLA locus or 8 DNA samples for multiple loci, is possible. Forty-five DNA samples were selected for HLA class I typing and were d derived from different DNA sources (blood, saliva and umbilical cord blood). Nine of these DNA samples had previously been identified as having novel alleles at one of their HLA class I loci using standard HLA typing methods. Whole gene amplicons were generated for the HLA class I genes. Each of the forty-five DNA samples were amplified for HLA-A, -B and -C using generic primers tagged with a DNA barcode unique to that sample resulting in 135 barcoded amplicons. Results Sufficient numbers of reads were obtained for each locus of each sample to enable HLA allele calling (mean 145; range 47–385) with mean a QV > 75. 252 HLA class I alleles were expected from the 45 DNA samples when homozygous loci were considered. The HLA types of 219 of these alleles immediately matched the types observed previously. The consensus sequences also confirmed the presence and phasing of the variants within the nine novel alleles. The consensus sequences on the 24 remaining alleles contained novel differences when compared to existing reference sequences in the IMGT/HLA Database. Sanger sequencing is being used to verify these novel positions. Conclusion SMRT DNA sequencing has the capability and the accuracy to facilitate definitive allele-level resolution HLA typing. We have shown for the first time that it is possible to use this method to sequence multiple DNA samples for multiple HLA class I genes using DNA barcode technology, to a level that makes it viable for routine use as a high-throughput methodology. The identification of previously unknown mismatches between recipients and their donors could significantly improve stem cell transplant outcome. S. Ranade: Employee; Company/Organization; Pacific Biosciences . K. Eng: Employee; Company/Organization; Pacific Biosciences. B. Bowman: Employee; Company/Organization; Pacific Biosciences . L. Hepler: Employee; Company/Organization; Pacific Biosciences.