High throughput, low cost detection of Gamma block SNPS using a capture probe target enrichment next generation sequencing assay

Abstract

Aim Previous studies have demonstrated that mismatching for SNPs in the Gamma block of the central MHC increases the risk of severe acute GvHD following unrelated HSCT. These studies were performed using PCR-SSP, a protocol that is not amenable to high throughput testing. In order to facilitate high throughput testing for retrospective studies we have developed a capture probe based next generation sequencing assay for the detection of Gamma block SNPs. Methods We designed 120 mer capture probes tiling the genetic region of interest. 200 ng of DNA from 20 samples that had previously been Gamma-Type™ tested by PCR-SSP were fragmented using a Covaris M220 Ultrasonicator™ using the following conditions: duty factor 20%, peak incident power 50 W, 200 cycles per burst, for 45 s at a temperature of 20 °C – to give a peak distribution of fragments approximately 550 bp in length. The fragments were repaired, size selected by a dual-bead based protocol, adenylated and adapters were ligated. The fragments were enriched and capture probes were used to isolate fragments containing Gamma block specific sequences. The Gamma block specific fragments were sequenced using a MiSeq® Next Generation DNA Sequencer, generating 300 bp paired-end reads. The Gamma-Type™ variant positions were annotated within Assign™ MPS resulting in automated reporting of Gamma block SNPs enabling direct comparison between donors and patients. Results Even coverage of > 1000 calls were obtained for all samples across all positions. There was complete concordance between the PCR-SSP results and the capture probe assay, with additional polymorphisms seen for two of the samples. The additional polymorphisms did not affect the matching/mismatching outcome with the donor/patient pairs. Conclusion The capture probe approach is amenable to low cost and high throughput testing with the added advantage that the precise sequence is reported, whereas by PCR-SSP, only the PCR target sequence is detected. Furthermore the Gamma block capture probes can be included with HLA probes to provide complete HLA and Gamma block SNP detection in one assay. Large retrospective studies have the potential to identify the non-HLA regions within the MHC that influence outcomes in unrelated HSCT.