P086 : IMPROVED ION TORRENT SEQUENCING CHEMISTRY ENABLES RAPID TURN AROUND AND 600BP READS

Abstract

Application of next generation sequencing (NGS) technologies to HLA sequencing has been a challenge due to the requirements of high accuracy, rapid process time and longer read lengths. Ion Torrent(TM) reagents currently support fragments up to ∼430 bp with basecalling accuracy on par with other platforms. We have evaluated the performance of new isothermal amplification (IA) reagents for clonal amplification of sequencing templates and Ion PGM(TM) Hi-Q(TM) sequencing reagents in the context of HLA sequencing. To investigate these new reagents we compared performance to the current Ion Torrent 400 bp chemistry using 120 samples over 4 PGM runs. The HLA libraries were prepared using a 400 bp HLA exon specific multiplex (Mx) PCR assay and a range of longer PCR products up to 630 bp in size. The MxPCR assay covered the antigen recognition exons of HLA-A, B, C, DRB1, DQB1, DPB1 and was used to evaluate typing concordance. The long amplicons were designed to create a contiguous PCR fragment including exon2 through intron2 and exon3 of HLA-B and C. These amplicons were used to evaluate the read length limit of the new chemistry. NGS library preparation of the PCR products followed the standard Ion Torrent adaptor ligation protocol. All adapted libraries were divided and run with the IA and Hi-Q reagents as well as the current 400 bp chemistry. The HLA TypeStream(TM) software was used for HLA typing and sequencing analysis. Using the MxPCR assay, typing results were equivalent between the chemistries ranging from 95% to 100% depending on the locus. The modal read length increased with the IA and Hi-Q chemistry from 306 to 319 bp. The long PCR amplicon results produced modal read lengths up to 602 bp and provided complete bidirectional sequence data from the amplicons. Homopolymer sequence accuracy showed improved or equivalent results using the new chemistry depending on the sample and length of the homopolymer. In this study, the new chemistry shows improved performance with longer read lengths and basecalling accuracy. The potential benefits of the IA and Hi-Q chemistry are improved HLA typing and ambiguity resolution. An additional benefit of the new chemistry is a reduction in process time by ∼8 h which offers the potential of a single day NGS workflow. D. Dinauer: Employee; Company/Organization; ThermoFisher. Stock Shareholder; Company/Organization; ThermoFisher. J. Bollmer: Employee; Company/Organization; ThermoFisher. Z. Chen: Employee; Company/Organization; ThermoFisher. C. Bialozynski: Employee; Company/Organization; ThermoFisher. J. Shi: Employee; Company/Organization; ThermoFisher. S. Conradson: Employee; Company/Organization; ThermoFisher. Stock Shareholder; Company/Organization; ThermoFisher.