Establishing a Next Generation Sequencing laboratory for high resolution HLA typing

Abstract

Aim To evaluate NGS (Next Generation Sequencing) platforms for high resolution HLA typing in a clinical setting. HLA mismatches can have a significant effect on transplantation outcome. Current low resolution HLA typing techniques are unable to provide coverage for the growing number of known alleles. Sanger sequencing, the standard for high resolution typing, cannot provide full gene coverage or phasing. Newly developed Next Generation Sequencing kits accomplish full length gene sequencing and allele phasing. NGS reduces ambiguity in HLA genotyping, providing the best possible results for transplant matching. Full gene sequencing also allows for the discovery of novel alleles. Method Two assay platforms are being evaluated. Whole genes are amplified by long range polymerase chain reaction, yielding amplicons ∼3–10 Kilobases in length. Long amplicons are randomly fragmented using proprietary enzymatic methods. Unique “barcode” sequence adapters are ligated to fragment ends. Fragments are combined to create a paired end sequencing library. Size selection is performed for best quality sequencing reads. Libraries are sequenced on the Illumina MiSeq (40 h). Analysis software assembles reads and assigns HLA typing based on IMGT (international ImMunoGeneTics project)/HLA database information. Results Our first sequencing run (12 samples – 96 loci) yielded 97% concordance. Data points were eliminated due to: no amplification or sequencing due to possible degraded DNA (4), novel variant (1), full reference sequence unknown (2), analysis errors since resolved with software development (6). Issues requiring troubleshooting include amplification failure at one locus and uneven sequence coverage for Class I loci. Additional sequencing runs will be analyzed and compared. Limitations of NGS include blocking of SNP positions due to primer placement and lack of full IMGT sequences of some alleles. Conclusion Workflow complexity, turnaround time, and cost of instrumentation and consumables must be considered in a decision to adopt NGS in a clinical HLA laboratory. Meticulous techniques which can impact outcome must be mastered. NGS is useful to sequence multiple samples using DNA barcodes which makes it viable for routine use, which may significantly improve transplantation outcome.