128-P: EVALUATION OF LONG READ SEQUENCING CHEMISTRY FOR NEXT GENERATION SEQUENCING ON THE PGM PLATFORM

Abstract

Next generation sequencing (NGS) technologies offer several advantages over current HLA genotyping methods including complete coverage of gene sequences and lower per sample costs due to the massively parallel sequencing of millions of fragments in a single run. Additionally, clonal amplification of template fragments during the template preparation process can enable cis/trans ambiguity resolution. Many common cis/trans ambiguities require read lengths over 300bases. We are developing an HLA typing assay on the Ion Torrent Personal Genome Machine® (PGM) platform and have compared the evolution of sequencing chemistries for this semiconductor sequencing platform. Long range PCR strategies were used to compare different read length reagents and protocols. Over 20 different samples have been re-sequenced for the HLA-A,B,C,DRB1 genes to evaluate the scalability of the platform. Sequence reads covering each gene were analyzed using in-house software tools and assessed for accuracy, read length, and cis/trans ambiguity resolution. The latest protocols provide superior results for all metrics. The most challenging typings were selected to compare sequencing chemistry for cis/trans phasing over exon2 and exon3 of class I genes. The 200bp Ion Torrent reagents show the longest perfect match read of 252 bases and a modal read length of 200 bases. More recent protocols produce long reads over 450 bases with modal read lengths over 350 bases. Phase ambiguity resolution was accomplished with both chemistries but the longer read methods produced more contiguous reads over the cis/trans region. The raw sequence accuracy improved from 96.6% to 97.2% using the updated protocols. The scalability of the PGM platform has been demonstrated by producing over double the sequence output while maintaining a high degree of raw read accuracy as protocols and reagents evolve. The current PGM protocols combined with full gene sequencing offers absolute HLA typing resolution in a single run. Zhao: Life Technologies: Employee. Bialozynski: Life Technologies: Employee. Shi: Life Technologies: Employee. Radick: Life Technologies: Employee. Conradson: Life Technologies: Employee. Dinauer: Life Technologies: Employee.