LBOR01: ONE MILLION SAMPLES TYPED BY NGS – LESSONS LEARNED

摘要: Aim DKMS Life Science Lab adopted an NGS based HLA typing workflow in early 2013. Meanwhile more than a million samples have been successfully typed by forming the basis for this review of chances and pitfalls such approach. Methods Our is on direct sequencing PCR amplified exons Illumina MiSeq instruments. Exons 2 3 HLA-A, B, C, DRB1, DQB1 DPB1 are Fluidigm Access Array chips. Recently profile was extended to cover addition ABO, Rhesus CCR5. Amplicons up 768 pooled run. Sequencing data analysed in-house developed software neXtype. Results The rather simple proved be very robust. Loading chips critical and, due low volumes, needs careful optimization regular maintenance involved liquid handlers. continuously yielded sufficient product loading sequencers. MiSeqs general produce high quality sequence data. Low diversity like amplicon products pose particular challenge but meanwhile handled well. However, care must taken keep rate short artefacts as those may negatively impact overall quality. For analysis identify crossover which resemble true alleles. In addition, error at position might significantly higher indicated q -values. When taking into account neXtype can provide highly automated accurate results fraction time Sanger analysis. Given adequate we identified imbalanced amplification main source error. Rigorous control every reagent batch with regard even essential. DNA concentrations below 3 ng/μl should not applied 192 avoid random loss Conclusions Switching primary tool beneficial lab. has proven superior previous aspect: Costs, hands time, resolution, turnaround stability scalability. We conclude that technology matured point where it appropriate everyday routine operation.