Using Oxford Nanopore and long-range PCR to establish a genotyping method for killer immunoglobulin-like receptor genes and their ligands encoded by human leukocyte antigen genes

Abstract

Background: The human leukocyte antigen (HLA) gene region is considered the most polymorphic region in the human genome and encodes for transmembrane HLA proteins present on nucleated and antigen presenting cells (APCs). HLA proteins are divided into classes I-III, wherein class I (HLA-A, -B and -C) and class II (HLA-DR, -DQ and -DP) proteins are clinically significant due to their binding and presentation of intra- or extracellular peptides. HLA-B and -C, as well as some -A alleles, are also ligands to the killer immunoglobulin-like receptors (KIRs) which are transmembrane proteins on natural killer (NK) cells. Both HLA and KIR are also implicated in several autoimmune diseases. Moreover, HLA and KIR are both complex gene regions, affected by polymorphisms. KIR are also challenging to sequence due to copy number variations (CNVs) and homology. As such, there are currently limited sequencing approaches for KIR, in contrast to HLA.

Aim: The aim of this thesis was to establish and optimize a combined genotyping method for KIR as well as HLA-A, -B and -C. This was attempted through developing cost-effective long -range (LR) multiplex PCR methods and a long-read sequencing technique.

Materials and methods: High molecular weight genomic DNA (HMW gDNA) (n=1) was extracted from peripheral blood mononuclear cells (PBMCs) and compared to pre-extracted gDNA (n=5) during amplification and sequencing. KIR and HLA class I genes were amplified in their own LR multiplex PCRs using different primers and polymerases. Self-designed primers amplifying the HLA-A, -B and -C genes including regulatory regions, were compared to published gene-amplifying HLA primers. KIRs were amplified using published primers. Amplicons were visualized through gel electrophoresis and automatic electrophoresis. Varying combinations of HLA and KIR amplicons in different libraries were sequenced on the Oxford Nanopore platform.

Results: There was no observed significant difference in amplicon length between the two DNA types. Self-designed HLA primers produced unspecific and short amplicons compared to published HLA primers. Additionally, the short KIR amplicons were preferentially amplified compared to long KIR amplicons. A favored sequencing of short reads such as short KIR was also observed. HLA-A reads were the most abundant in each library, outperforming HLA-B and -C.

Conclusion: While it was possible to amplify and sequence the HLA and KIR genes to a certain degree, it was not possible to develop a combined genotyping method for KIR and HLA under the scope of this thesis. In conclusion, further development and optimization is needed to even out the imbalance of short amplicon and read preference present during amplification and sequencing.