Vinzenz Lange

Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans

Mass-spectrometry-based methods for relative proteome quantification have broadly affected life science research. However, important research directions, particularly those involving mathematical modelling and simulation of biological processes, also critically depend on absolutely quantitative data—that is, knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a considerable fraction of the proteome (73%) have only been derived from genetically altered Saccharomyces cerevisiae cells, a technique that is not directly portable from yeast to other species. Here we present a mass-spectrometry-based strategy to determine the absolute quantity, that is, the average number of protein copies per cell in a cell population, for a large fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for leptospirosis, we generated an absolute protein abundance scale for 83% of the mass-spectrometry-detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo-electron tomography morphological measurements to verify, at the single-cell level, the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. Because the strategy is relatively fast and applicable to any cell type, we expect that it will become a cornerstone of quantitative biology and systems biology.

Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing

BackgroundA close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.ResultsWe have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed “neXtype” for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.ConclusionsThe workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

High-resolution HLA haplotype frequencies of stem cell donors in Germany with foreign parentage: How can they be used to improve unrelated donor searches?

In hematopoietic stem cell transplantation, human leukocyte antigens (HLA), usually HLA loci A, B, C, DRB1 and DQB1, are required to check histocompatibility between a potential donor and the recipient suffering from a malignant or non-malignant blood disease. As databases of potential unrelated donors are very heterogeneous with respect to typing resolution and number of typed loci, donor registries make use of haplotype frequency-based algorithms to provide matching probabilities for each potentially matching recipient/donor pair. However, it is well known that HLA allele and haplotype frequencies differ significantly between populations. We estimated high-resolution HLA-A, -B, -C, -DRB1 haplotype and allele frequencies of donors within DKMS German Bone Marrow Donor Center with parentage from 17 different countries: Turkey, Poland, Italy, Russian Federation, Croatia, Greece, Austria, Kazakhstan, France, The Netherlands, Republic of China, Romania, Portugal, USA, Spain, United Kingdom and Bosnia and Herzegovina. 5-locus haplotypes including HLA-DQB1 are presented for Turkey, Poland, Italy and Russian Federation. We calculated linkage disequilibria for each sample. Genetic distances between included countries could be shown to reflect geography. We further demonstrate how genetic differences between populations are reflected in matching probabilities of recipient/donor pairs and how they influence the search for unrelated donors as well as strategic donor center typings.

2.7 million samples genotyped for HLA by next generation sequencing: lessons learned

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.

ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing

BackgroundThe characterization of the ABO blood group status is vital for blood transfusion and solid organ transplantation. Several methods for the molecular characterization of the ABO gene, which encodes the alleles that give rise to the different ABO blood groups, have been described. However, the application of those methods has so far been restricted to selected samples and not been applied to population-scale analysis.ResultsWe describe a cost-effective method for high-throughput genotyping of the ABO system by next generation sequencing. Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. We report the allele group frequencies calculated on a subset of more than 110,000 sampled individuals of German origin. Further we discuss the potential of the workflow for high resolution genotyping taking the observed allele group frequencies into account. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples.ConclusionsThe described workflow delivers high resolution ABO genotyping at low cost enabling population-scale molecular ABO characterization.

Prediction of spurious HLA class II typing results using probabilistic classification.

While modern high-throughput sequence-based HLA genotyping methods generally provide highly accurate typing results, artefacts may nonetheless arise for numerous reasons, such as sample contamination, sequencing errors, read misalignments, or PCR amplification biases. To help detecting spurious typing results, we tested the performance of two probabilistic classifiers (binary logistic regression and random forest models) based on population-specific genotype frequencies. We trained the model using high-resolution typing results for HLA-DRB1, DQB1, and DPB1 from large samples of German, Polish and UK-based donors. The high predictive capacity of the best models replicated both in 10-fold cross-validation for each gene and in using independent evaluation data (AUC 0.820-0.893). While genotype frequencies alone provide enough predictive power to render the model generally useful for highlighting potentially spurious typing results, the inclusion of workflow-specific predictors substantially increases prediction specificity. Low initial DNA concentrations in combination with low-volume PCR reactions form a major source of stochastic error specific to the Fluidigm chip-based workflow at DKMS Life Science Lab. The addition of DNA concentrations as a predictor variable thus substantially increased AUC (0.947-0.959) over purely frequency-based models.

Frequencies of gene variant CCR5-Δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national DKMS donor centers

Homozygous carriers of CCR5-Δ32, a gene variant of CC-type chemokine receptor 5 (CCR5), are highly resistant to infections with human immunodeficiency virus type 1 (HIV-1) and therefore preferred stem cell donors for HIV-infected patients. We analyzed CCR5 typing data of 1,333,035 potential hematopoietic stem cell donors enlisted with three national DKMS donor centers. Allele and genotype frequencies were determined for 87 countries of origin as self-assessed by the donors. CCR5-Δ32 allele frequencies ranged from 16.4% in the Norwegian sample to 0 in donors from Ethiopia. The highest CCR5-Δ32/Δ32 genotype frequency was found in the sample from the Faroe Islands (2.3%), whereas in 27 samples, predominantly of donors from Africa, Asia and South America, none of the individuals carried this genotype. The characteristic CCR5-Δ32 allele frequency decline from Northern to Southeastern Eurasia supports findings of earlier studies. With available HLA haplotype frequency information for the patients ethnicity, our data allows upfront estimation of the probability that an HLA-matched donor with CCR5-Δ32/Δ32 genotype can be found for a patient in need of hematopoietic stem cell transplantation.

P070 A novel NGS workflow reveals MICA ALLELE frequencies based on 350,000 German samples

Aim The MICA and MICB molecules serve as ligands of the activating NKG2D receptor expressed on natural killer (NK) cells. Recent studies indicate effects of MIC genotypes on hematopoietic stem cell transplantation (HSCT) outcome. To provide MIC allele information for donor selection, we developed an NGS-based high-throughput genotyping workflow for MICA and MICB and applied it for genotyping registry donor samples. Methods Exons 2 and 3, as well as most of exons 4 and 5 of MICA and MICB are amplified in a multiplexed PCR reaction. The PCR products are sequenced on Illumina HiSeq or MiSeq instruments. The data are processed by an updated neXtype software version to provide allele-level genotyping information. Results Using this NGS based workflow, we genotyped 350,000 donors registered in Germany and report on the observed MICA allele frequencies. Due to the restricted sequence coverage, 9 alleles encoding distinct proteins cannot be resolved. However, the unprecedented depth of the study allowed us to estimate allele frequencies for 49 of the 84 described MICA alleles distinguished at the protein coding level. In addition we identified novel alleles in 0.2% of the samples. The 13 (31) most abundant alleles account for a cumulative allele frequency of 99% (99.99%). Conclusions This newly developed NGS-based genotyping approach offers the opportunity to analyze the genetic diversity of MICA and MICB in large cohorts at high-resolution.

P103 The promise of cost-efficient full-length HLA class I genotyping: Advances using nanopore sequencing

Aim Nanopore based sequencing has seen rapid advancement in the recent years with iterations in pore structure, sequencing chemistries and base callers. This has led to increasingly accurate sequencing of extremely long DNA molecules. Since current HLA genotyping algorithms are not optimized for nanopore data we developed a genotyping algorithm based on read grouping and subsequent mapping to a reduced reference database. Methods High raw sequencing errors present a challenge for genotyping based on direct mapping to a reference database. Our algorithm, Poretyper, obviates this initial mapping by first grouping the raw reads based on the distributions of k-mers within each read. A multiple sequence alignment derived from the groups of raw reads results in a set of consensus sequences which represent potential alleles. These consensus sequences are then used to create a culled reference database dramatically reducing the search space, thus reducing artefactual raw read mappings. Results HLA-A, HLA-B and HLA-C for a hundred samples drawn from the DKMS donor registry were sequenced using the MinION with R9.5 chemistry. All hundred samples were then genotyped using Poretyper and existing G-group pretypings could be recapitulated failing only for those alleles where no full length sequences were available. Conclusions Nanopore sequencing presents a viable and accurate platform for cost-efficient full-length HLA Class I genotyping. For those alleles where full length reference sequences are not available, an in silico extension of such allele sequences using the full-length sequence of the next closest allele presents a viable approach for full-length genotyping.

P091 Typeloader for windows: Automated full length submission of novel alleles

Aim Many of the alleles submitted to the IPD-IMGT/HLA and IPD-KIR databases are restricted to the coding sequence or even, for HLA, to the exons encoding the antigen recognition domain. This impedes the establishment of long-read-based genotyping technologies. It is desirable that novel alleles are characterized and submitted in full length, and that known alleles are extended to cover the complete gene sequence. However, the manual annotation and submission of full-length sequences to the IPD-IMGT/HLA and ENA databases is a time-consuming and error-prone task. In 2016, we developed and published TypeLoader, a tool that takes the full-length sequence of a novel HLA allele in FASTA format as input, automatically annotates it, and creates all files necessary for submission. This reduced the manual effort for submission by over 95%. It was implemented as a web application to run on a Linux server and has been used at the DKMS Life Science Lab to successfully submit more than 1000 novel HLA alleles. Methods To enable a more widespread use of TypeLoader by other labs, we have adapted the tool to make it available as a Windows standalone application. We replaced the server-dependent web form with a convenient GUI (graphical user interface) using Python3 and PyQt5, which support most common operating systems. An internal SQLite database was added to store a wide range of metadata about each allele, which are accessible via the GUI. As a further improvement, TypeLoader now automatically detects null alleles generated by premature stop codons or frameshift mutations, and features enhanced integration with ENA’s automated submission API. TypeLoader also implements experimental support for annotation and submission of KIR alleles. Results The new TypeLoader can easily be used on standard PCs without dependencies on other software. With the added internal database, TypeLoader can be used as a lab’s central information platform on their full-length sequences meant for submission. Conclusions We hope that the increased convenience and scope of TypeLoader will foster the submission of more full-length sequences to the IPD-IMGT/HLA and IPD-KIR databases, ultimately promoting the widespread use of full-length sequencing for genotyping of both HLA and KIR.