BackgroundAt the DKMS Life Science Lab, Next Generation Sequencing (NGS) has been used for ultra-high-volume high-resolution genotyping of HLA loci for the last three and a half years. Here, we report on our experiences in genotyping the HLA, CCR5, ABO, RHD and KIR genes using a direct amplicon sequencing approach on Illumina MiSeq and HiSeq 2500 instruments.ResultsBetween January 2013 and June 2016, 2,714,110 samples largely from German, Polish and UK-based potential stem cell donors have been processed. 98.9% of all alleles for the targeted HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) were typed at high resolution or better. Initially a simple three-step workflow based on nanofluidic chips in conjunction with 4-primer amplicon tagging was used. Over time, we found that this setup results in PCR artefacts such as primer dimers and PCR-mediated recombination, which may necessitate repeat typing. Split workflows for low- and high-DNA-concentration samples helped alleviate these problems and reduced average per-locus repeat rates from 3.1 to 1.3%. Further optimisations of the workflow included the use of phosphorothioate oligos to reduce primer degradation and primer dimer formation, and employing statistical models to predict read yield from initial template DNA concentration to avoid intermediate quantification of PCR products. Finally, despite the populations typed at DKMS Life Science Lab being relatively homogenous genetically, an analysis of 1.4 million donors processed between January 2015 and May 2016 led to the discovery of 1,919 distinct novel HLA alleles.ConclusionsAmplicon-based NGS HLA genotyping workflows have become the workhorse in high-volume tissue typing of registry donors. The optimisation of workflow practices over multiple years has led to insights and solutions that improve the efficiency and robustness of short amplicon based genotyping workflows.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3575-z) contains supplementary material, which is available to authorized users.

The genetically and antigenically diverse group of noroviruses is the major cause of human viral epidemic gastroenteritis worldwide. Virus detection and control are thus crucial topics when aiming at containing and preventing the resulting large and often persisting outbreaks. Aptamers provide a promising alternative to antibodies concerning their ability to bind and thus detect and influence bio-active molecules. These small, single-stranded oligonucleotides are able to bind to a multitude of possible target molecules with high affinity. For a specific target the highest affinity aptamers are found by screening a randomized library. In this work a DNA aptamer capable of binding to the norovirus genotype II.4 capsid protein VP1 was found. The general approach is thereby not limited to norovirus capsid, but could be extended to almost any kind of biologically relevant molecule. The development of the library enrichment was further computationally analyzed in order to describe the enrichment during screening. This is the basis for a later extensive characterization of both target and aptamers that could lead to insights regarding the functional coherence of both partners. An abstract model describing this coherence could be utilized to generate a target-specific library, from which future aptamer screening runs could benefit.

Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunologic compatibility between organ donors and recipients, but the platform's high error rate makes it challenging to type alleles with accuracy. We developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database. Using two independent data sets generated on the R9.4 flow cell chemistry, Athlon achieved a 100% accuracy in class I HLA typing at the two-field resolution.

Aptamers are synthetic single-stranded oligonucleotides which bind specifically to their target. They offer several advantages over antibodies. For example, aptamers can be produced under unphysiological conditions against almost any target, including toxic or pathological substances. They are also quicker and cheaper produced than antibodies, and are easy to modify without loss of activity. Furthermore, they exhibit high stability under a width range of conditions. Consequently, they make excellent receptors for the use in biosensors. This article describes the evaluation of a novel aptasensor based on the Surface Plasmon Resonance (SPR)-system developed by the Fraunhofer Institute for Applied Optics and Precision Engineering IOF (Jena, Germany) using a thrombin–aptamer interaction as a model system. The biotin-tagged aptamer was attached to the sensor's gold surface by means of its interaction with streptavidin. Thrombin solutions of different concentrations were pumped over this surface, and the interaction was measured under buffer flow. The binding signals for the thrombin–aptamer interaction were compared to those arising from a control random-oligonucleotide of the same size and bearing the same modifications. Using this approach, we were able to obtain reproducible, significant and stable signals with a limit of detection of about 26 nmol/L

Nanopore sequencing, a paradigm change in sequencing technologies, offers a new cost-effective and scalable platform for HLA genotyping. Among the new generation of high-throughput sequencing technologies, the MinION nanopore sequencer is the first to offer a non-template-based direct DNA sensing sequencing technology. Oxford Nanopore Technologies (ONT) introduced the first version of the MinION in 2014; since then, the platform has gone through multiple iterations resulting in higher throughput and sequencing accuracy. The "what you put in is what you get" nature of the platform enables molecules to be sequenced without fragmentation. This results in ultra-long read lengths in the order of tens of kilobases enabling entire genes to be characterized with fully phased sequence information. With release R9.5, the MinION platform has reached a quality that enables HLA genotyping with minor shortcomings in long homopolymer regions. Within this chapter, we describe a protocol for sequencing and genotyping HLA Class I alleles using the MinION.