Diagnostic Applications of Next Generation Sequencing in Immunogenetics and Molecular Oncology

Abstract

With the introduction of the next generation sequencing (NGS) technologies, remarkable new diagnostic applications have been established in daily routine. Implementation of NGS is challenging in clinical diagnostics, but definite advantages and new diagnostic possibilities make the switch to the technology inevitable. In addition to the higher sequencing capacity, clonal sequencing of single molecules, multiplexing of samples, higher diagnostic sensitivity, workflow miniaturization, and cost benefits are some of the valuable features of the technology. After the recent advances, NGS emerged as a proven alternative for classical Sanger sequencing in the typing of human leukocyte antigens (HLA). By virtue of the clonal amplification of single DNA molecules ambiguous typing results can be avoided. Simultaneously, a higher sample throughput can be achieved by tagging of DNA molecules with multiplex identifiers and pooling of PCR products before sequencing. In our experience, up to 380 samples can be typed for HLA-A, -B, and -DRB1 in high-resolution during every sequencing run. In molecular oncology, NGS shows a markedly increased sensitivity in comparison to the conventional Sanger sequencing and is developing to the standard diagnostic tool in detection of somatic mutations in cancer cells with great impact on personalized treatment of patients.

Are polymorphisms of the immunoregulatory factor CD40LG implicated in acute transfusion reactions?

“…On the other hand, this technical strategy could be used for HIGM1 investigation (we covered 182 of all the 183 mutations in CD40LG that cause HIGM1 (http://www.hgmd.org/ updated in June 2014). It is less expensive than actual used techniques, especially in developing countries such as Tunisia where HIGM syndrome is frequently found466162.…”

Conflicting HLA assignment by three different typing methods due to the apparent loss of heterozygosity in the MHC region

“…However, in both studies, Sanger sequencing was the least sensitive method, probably because of its specific sequencing primers in contrast to the more generic primers used in SSP and rSSO. It remains to be seen how the new HLA kits based on the next generation sequencing techniques will unravel LOH, as less than 1.5%–10% of the background sequence can be assigned to another allele by these new sequencing techniques .…”

HLA‐B*44:138Q: Evidence for a confined deletion and recombination events in an otherwise unaffected HLA‐haplotype

“…In summary, the characterisation of HLA‐B*44:138Q on the IonTorrent platform allowed a fast analysis of whole genes with much less effort compared with Sanger sequencing. However, the homopolymer issue of NGS remains a diagnostic challenge. The generation of the new allele within a haplotype that otherwise consists of common HLA‐alleles (as assessed by full length sequencing) indicates that the mechanisms leading to this new allele are restricted to a single gene.…”