A Comparative Study Of Hla Typing Using An Illumina Miseq Ngs System.

Although the Luminex system for HLA typing provides a convenient, easy to perform laboratory workflow, limitations restricting the ability to routinely obtain both breadth (number of loci) and depth (allele resolution) of data are inherent in the system. If high-resolution data is required, high volume laboratories can minimize costs effectively but for clinical laboratories in direct support of hematopoetic cell transplants (HCTs) costs of dye-terminator sequence-based typing are relatively high. In order to improve the economy of obtaining both broad and high-resolution data for our laboratory, we tested a next generation sequencing (NGS) method for HLA typing. The method uses the Illumina MiSeq device combined with commercial software providing a convenient workflow for data generation and analysis. Data for HLA-A, B, C, exons 1-7 are obtained with phase determined by a combination of overlapping sequences and database lookup. Data for DRB1/3/4/5, DQA1, DQB1, DPA1, and DPB1 are obtained from exons 1-4 with phase between exons determined by coincidence with established databases. This approach was tested with parallel typing using the NGS and Luminex systems for over 200 samples to date. We intend to continue with parallel typing during the next few months for a total of about 500 samples for our final report. Overall results demonstrated unambiguous 3-field (six-digit) typing for all HLA class I and II with less than 1% failure rate and complete coincidence with Luminex data. Improvements in both accuracy and resolution are provided by the NGS system, and laboratory effort required is similar. Software and data storage are performed on the cloud, providing rapid analysis, ease of use, and freedom from on site systems support. We detail the comparative typing results and critically examine the NGS workflow towards establishing the efficacy and economy of this NGS approach in laboratories supporting HCTs.