P098 : HLA TYPING USING ION TORRENT PGM AND LONG RANGE HLA GENE AMPLIFICATION PRIMERS: EVALUATION OF ISOTHERMAL AMPLIFICATION AND HI-Q CHEMISTRY

Abstract

Aim The goal of this study is to evaluate and optimize the Isothermal Amplification (IA) combined with Hi-Q sequencing chemistry, a new clonal amplification workflow for Ion Torrent PGM, for HLA typing to improve the data quality and typing accuracy. Method The testing panel consisted of 8–48 reference samples with diversified HLA typing including purified DNA from cultured cell lines and clinical samples derived from buccal swab. Samples were amplified using long range and mid-range HLA gene amplification primers. The amplicons were processed according to Ion Torrent PGM sequencing workflow using fragmentation and barcode attachment, followed by emulsion PCR as a control and IA workflow, and PGM sequencing. Results The initial results using 314 and 316 chips showed the longest read length of approximately 580 bp, increased coverage and improved uniformity in the coverage. Clonality was slightly higher in the range of 39 to 44%. The clonal amplification step has been reduced from 8 h to 90 min. HLA typing accuracy was comparable to that of emulsion PCR workflow. Better workflow using more practical parameters for routine HLA typing are being investigated. Conclusion IA is a rapid emulsion-free clonal amplification technology, which amplifies library templates on Ion Sphere Particles for PGM sequencing. In this work, we demonstrated the potential of IA and Hi-Q chemistry including: (1) longer read length of up to 600 bp, (2) significantly faster template preparation time of ∼2 h instead of ∼10 h for emulsion PCR, (3) improved homopolymer identification, (4) significant improvement in coverage uniformity. Our initial test results showed that IA +Hi-Q is an effective alternative to emulsion PCR with great potential to provide a powerful workflow for HLA typing. K. Saito: Employee; Company/Organization; Thermo Fisher. K. Beutner: Employee; Company/Organization; Thermo Fisher. D. Berman: Employee; Company/Organization; Thermo Fisher. O. Sanchez: Employee; Company/Organization; Thermo Fisher Scientific. Z. Chen: Employee; Company/Organization; Thermo Fisher. M. Wei: Employee; Company/Organization; Thermo Fisher. J. Lee: Employee; Company/Organization; Thermo Fisher.