MinION Single Molecule Sequencing: the new way of HLA allele resolution typing in low and high volume laboratories

M Groeneweg, C.E. Voorter, T.W.M. Slangen, C.M. Meertens, F. Palusci, M.G.J. Tilanus

Chair(s): Drs. K.C.A. Geneugelijk & prof. dr. C. van Kooten

Thursday 9 march 2017

15:30 - 15:42h at Hendrik Marsmanzaal

Categories: Parallel - Basaal

Parallel session: Parallelsessie XV - Basaal - Moleculaire analyses en het adaptieve immuunsysteem in orgaantransplantatie

The Human Leucocyte Antigen (HLA) complex plays a major role in the defense against foreign pathogens. In stem cell transplantation the highest possible resolution matching of patient and donor increases the chance of a successful outcome. In organ transplantation the detection of HLA antibodies against HLA epitopes urges HLA typing at the allelic level for patient and donor, especially in highly immunized patients. Allelic resolution HLA typing has evolved from PCR and probe based techniques to Sanger sequencing  and Next Generation Sequencing nowadays applied in many HLA laboratories across the world. These techniques are rather expensive (Sanger sequencing) and/or time consuming (NGS). We have now developed the MinION single molecule sequencing for HLA class I and class II. This method is a cheap sequencing method, with sample preparation and sequencing taking only a few hours and it can be used by both low and high throughput laboratories. With this application ultra-long reads are created, up to 100,000 bases, which enables the production of reliable fully phased stretches of full length HLA sequences in contrast to NGS methods with short reads. Results from class I amplicon based MinION sequencing confirmed the reliability,  validity and efficiency of the method with sufficient read depth, covering the full length gene from the start of the 5’ UTR to the end of the 3’ UTR. Initial results from class II full length sequencing already shows the applicability of this method for DQA1, DQB1, DPA1 and DPB1 with one amplicon, whereas for full length DRB1/3/4/5 two separate amplicons are needed due to the length of the gene. At the moment we are investigating the recently described probe captured method for the complete HLA region in combination with MinION sequencing, which would make the allelic resolution typing of deceased donors feasible within the limited time frame.