Aim: Enhancements in Next Generation Sequencing (NGS) library preparation, product performance, and typing resolution have enabled adoption of this technology for HSCT and SOT applications by clinical laboratories. Typical enrichment of HLA targets involves long range-PCR. However, an alternative method such as hybrid capture offers flexible and comprehensive gene coverage. A simple and robust 19 loci hybrid capture NGS assay was developed and optimized to achieve higher quality data with low quality samples. Performance of the 19 loci hybrid capture NGS assay was evaluated in a multi-center study consisting of three US HLA laboratories that currently use commercial NGS products in a clinical setting.
Method: 190 samples from whole blood or buccal swabs were used for evaluation. Genomic DNA samples were extracted with the QIAGEN EZ-1 or QIAsymphony. Genomic libraries for all samples were generated by fragmentation, adapter ligation, and library PCR. The resulting libraries were used for enrichment of 19 loci by a hybrid capture method. This process utilized biotinylated probes designed for 19 loci, followed by a streptavidin bead capture and a series of washes. A PCR reaction was performed to further enrich captured targets. The libraries were then sequenced on the Illumina MiSeq or iSeq instruments and fastq files were analyzed for genotyping assignments.
Results: The 19 loci hybrid capture NGS assay evaluated at three HLA laboratories produced good Quality Control metrics on two different Illumina instruments. Genotyping results showed more than 99% accuracy compared to typing from commercial NGS products and a reduced number of ambiguities. Improvements in allele balance for allele groups that are difficult with mainstream NGS assays were observed, including DRB1*04, DQB1*02/03 and DQA1*01 alleles. More than 60% of the reads mapped to the 19 loci targets, which resulted in an average read depth above 100x for 95% of the samples. Buccal swab-derived DNA, that previously failed with a commercial NGS assay, produced accurate typing results.
Conclusion: The 19 loci hybrid capture NGS assay produced accurate and robust results from the 3 clinical laboratories the study was performed in.
