P079 Experience using the illumina HLA trusight NGS kits: Version 1 vs. version 2

Abstract

We present the evaluation of two NGS-based HLA typing kits from Illumina, comparing TruSight kit version 1 with the newly released version 2 kit. Following Illumina’s protocol, 26 clinical and two IHWG DNA samples were evaluated using both kits. Samples had previously been Sanger sequenced. Pooled libraries were run on the Illumina MiSeq. Version 1 required the use of the 2 × 250 (500 cycle) MiSeq Reagent Kit whereas, version 2 required the use of the 2 × 150 (300 cycle) kit. Fastq files were analyzed using Conexio’s Assign software, versions 1.0.0.729 and 2.0.0.889 respectively. The version 2 kit outperformed the version 1 kit in several respects: there were fewer failures, fewer two-field ambiguities for B and DRB1 genes, and fewer problems with coverage and quality. Improvements in typing resolution are due to changes in primer locations. In addition, changes to the library preparation procedure simplified the process. In version 2, sample amplicons are now pooled much earlier in the process resulting in less pipetting and fewer consumables required. The change from the 500 to 300 cycle MiSeq kit resulted in time savings. The 500 cycle kit required 40 h of instrument run time while the 300 cycle kit only required 20 h of run time. In version 1, 88.9% of all samples typed correctly and unambiguously (2-field) whereas 97.8% of samples did so in version 2. The improvements and change to the 300 cycle reagent kit enabled us to take six samples from genomic DNA to high-resolution HLA genotyping results in 48 h. Illumina has made numerous improvements to their HLA TruSight HLA NGS kit which has made it a viable option for clinical implementation.