Human leukocyte antigen matching at allelic resolution is proven clinically significant in hematopoietic stem cell transplantation, lowering the risk of graft-versus-host disease and mortality. However, due to the ever growing HLA allele database, tissue typing laboratories face substantial challenges. In light of the complexity and the high degree of allelic diversity, it has become increasingly difficult to define the classical transplantation antigens at high-resolution by using well-tried methods. Thus, next-generation sequencing is entering into diagnostic laboratories at the perfect time and serving as a promising tool to overcome intrinsic HLA typing problems. Therefore, we have developed and validated a scalable automated HLA class I and class II typing approach suitable for diagnostic use.

Background: Cutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa (RDEB), which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined. Methods: MiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression or knock-out of a dysregulated miRNA was assessed in migration and 3D-spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets. Results: Several miRNAs were identified as dysregulated in cSCCs compared to control skin. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2. Conclusion: The discovery that miR-10b mediates an aspect of cancer stemness – that of enhanced tumor cell adhesion, known to facilitate metastatic colonization – provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor–recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.

Next-generation sequencing (NGS) has changed genomics significantly. More and more applications strive for sequencing with different platforms. Now, in 2012, after a decade of development and evolution, NGS has been accepted for a variety of research fields. Determination of sequencing errors is essential in order to follow next-generation sequencing beyond research use only. This study describes the overall 454 system performance of using multiple GS Junior runs with an in-house established and validated diagnostic assay for human leukocyte antigen (HLA) exon sequencing. Based on this data, we extracted, evaluated and characterized errors and variants of 60 HLA loci per run with respect to their adjacencies. We determined an overall error rate of 0.18% in a total of 118,484,408 bases. 31.3% of all reads analyzed (n=349,503) contain one or more errors. The largest group are deletions that account for 50% of the errors. Incorrect bases are not distributed equally along sequences and tend to be more frequent at sequence ends. Certain sequence positions in the middle or at the beginning of the read accumulate errors. Typically, the corresponding quality score at the actual error position is lower than the adjacent scores. Here we present the first error assessment in a human next-generation sequencing diagnostics assay in an amplicon sequencing approach. Improvements of sequence quality and error rate that have been made over the years are evident and it is shown that both have now reached a level where diagnostic applications become feasible. Our presented data are better than previously published error rates and we can confirm and quantify the often described relation of homopolymers and errors. Nevertheless, a certain depth of coverage is needed, in particular with challenging areas of the sequencing target. Furthermore, the usage of error correcting tools is not essential but might contribute towards the capacity and efficiency of a sequencing run.