Target enrichment using capture probes: The future of HLA typing by next generation sequencing?

Aim Most target enrichment (TE) procedures for high resolution HLA typing by Next Generation sequencing have utilized full, or part, gene PCR amplification. This is in contrast to many other molecular diagnostic applications where target enrichment is performed using capture probes (CP). One possible reason why TE using CP hasn’t been attempted for HLA is the high level of polymorphisms found in some exons and concern that all existing and undescribed alleles won’t be detected. We have designed a probe set for HLA-A, B, C, DRB1, DRB3,4,5 and DQB1 that eliminates this concern. TE by CP has many advantages over PCR including higher throughput, fewer artefacts, lower costs and the ability to include additional genes without increasing work load. Methods 200 ng of Genomic DNA from 16 individuals with known HLA genotypes were fragmented using a Covaris M220 Ultrasonicator™ using the following conditions: duty factor 20%, peak incident power 50W, 200 cycles per burst, for 45 s at a temperature of 20 °C – aiming for a peak distribution of fragments 550 bp in length. The fragments were then repaired, size selected by a dual-bead based protocol, adenylated and adapters were ligated. The fragments were enriched and Capture-HLA™ probes for multiple loci were used to isolate fragments containing HLA specific sequences. The HLA specific fragments were further enriched and 300 bp paired-end reads were then sequenced using a MiSeq® Next Generation DNA Sequencer. The resulting data was then analysed using the Assign™ MPS sequence analysis software. Results Successful genotyping was obtained for all loci for all samples, including exons 2 and 3 for class I and exon 2 for class II genes. There was even allele balance across all genes. Potential novel alleles were also detected. Conclusions Validation of this approach is on-going. However preliminary data suggests that the use of CP has the potential to revolutionize HLA typing by providing inexpensive high resolution, high volume HLA typing, with the ability to include additional genes.