137-P: SINGLE MOLECULE REAL-TIME SEQUENCING OF FULL LENGTH HLA CLASS I GENES – THE PROMISE AND CURRENT REALITY

Aim To sequence the full length of HLA Class I genes using single molecule real-time (SMRT ® ) sequencing technology. Methods Two samples were amplified by Long-Range PCR at HLA-A, B, C loci. Full lengths of these genes (∼3.2 kb) were generated with custom designed, barcoded primers targeting 5’UTR and 3’UTR. DNA libraries were prepared by ligating SMRTbell™ adaptors to the HLA amplicons. This produced closed-circular structures consisting of a double-stranded insert (full length HLA amplicon), and a single-stranded hairpin loop (SMRTbell™) on either end. Sequencing primer was annealed to the open/single-stranded portion of the hairpin adaptor. Streptavidin-bound DNA polymerase was added to the primer-template complex enabling the binding of the template to the bottom of nano-sized wells on the SMRT Cell. DNA synthesis of fluorescently labeled nucleotides was observed in real-time on the PacBio ® RS instrument. The pooled libraries were run on 6 SMRT Cells using either 45 or 90 minute data collection time per SMRT Cell. Individual sequence reads ⩾1,000 bases were parsed by sample-specific barcode sequence and were then aligned using BWA-SW. Alignment data were analyzed with SAM Tools and custom written code. Results Sequence and Alignment Statistics: [ Table 1 ]. Conclusions We demonstrate real-time sequencing of HLA genes from single DNA molecules. This technology offers great potential for HLA genotyping and further characterization of the MHC due to the length and phase of sequences obtained, speed, and ease of sample prep. Ranade: Pacific Biosciences Inc.: Employee. Sethuraman: Pacific Biosciences Inc.: Employee. Chin: Pacific Biosciences Inc.: Employee. Robinson: Pacific Biosciences Inc.: Employee.