Aim: NGS instruments continue to evolve with significant improvements in data quality, run times, and costs. Such improvements enable adoption of this technology for routine typing of HLA and Non-Classical MHC loci for HSCT and SOT applications. Most NGS assays utilize LR-PCR or Hybrid Capture methods for the enrichment of classical HLA and additional gene targets. NGS assays have yet to be evaluated on the newest Illumina-MiSeq i100 instrument. In this study, we assessed compatibility of three NGS assays on the MiSeq i100 by monitoring sequencing data and accuracy across all targeted loci.
Methods: Twenty-four DNA samples from the Coriell Institute with reference types for HLA and Non-Classical MHC loci were used for evaluation. NGS libraries were prepared according to manufacturer instructions. For LR-PCR assays, the 11 Classical HLA genes were amplified and sequenced while the Hybrid Capture assay enabled enrichment of 19 Loci, including the 11 Classical HLA genes, with proprietary probes. Final NGS libraries were sequenced separately using a 5M Flow Cell (2 x 150bp read length) at a final concentration range of 100-125pM. Fastq files were analyzed on the TypeStream™ Visual Software 3.2 for final genotypic assignments.
Results: LR-PCR assays produced HLA amplicons with concentrations ranging from 26-55ng/uL for library preparation. The Hybrid Capture assay utilized genomic DNA inputs ranging from 140-400ng for library preparation and subsequent enrichment of 19 Loci. Resulting NGS libraries showed average fragment sizes ranging from 610-914bp on TapeStation. The MiSeq i100 produced high quality reads with an average %Q30 of 97% and more than 12M paired end reads per run in less than 8 hrs. Typing accuracy for NGS assays was 100% for all loci at Field-3 and Field-2 (MIC genes). LR-PCR assays showed coverage > 100 and allele balance > 0.3 in key exons for 11 Loci. The Hybrid Capture assay showed coverage > 100 and allele balance > 0.3 in all exons for 19 Loci.
Conclusion: The MiSeq i100 instrument produced accurate and high-quality data for three NGS assays with faster run times enabling sample to result in a single day.
