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 patient's 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.

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

BACKGROUND: At 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. RESULTS: Between 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. CONCLUSIONS: Amplicon-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.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: The described workflow delivers high resolution ABO genotyping at low cost enabling population-scale molecular ABO characterization.

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

BACKGROUND: A 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. RESULTS: We 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. CONCLUSIONS: The 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.

Regional HLA differences in Poland and their effect on stem cell donor registry planning.

Regional HLA frequency differences are of potential relevance for the optimization of stem cell donor recruitment. We analyzed a very large sample (n = 123,749) of registered Polish stem cell donors. Donor figures by 1-digit postal code regions ranged from n = 5,243 (region 9) to n = 19,661 (region 8). Simulations based on region-specific haplotype frequencies showed that donor recruitment in regions 0, 2, 3 and 4 (mainly located in the south-eastern part of Poland) resulted in an above-average increase of matching probabilities for Polish patients. Regions 1, 7, 8, 9 (mainly located in the northern part of Poland) showed an opposite behavior. However, HLA frequency differences between regions were generally small. A strong indication for regionally focused donor recruitment efforts can, therefore, not be derived from our analyses. Results of haplotype frequency estimations showed sample size effects even for sizes between n≈5,000 and n≈20,000. This observation deserves further attention as most published haplotype frequency estimations are based on much smaller samples.

High-resolution human leukocyte antigen allele and haplotype frequencies of the Polish population based on 20,653 stem cell donors.

We present high-resolution allele and haplotype frequency (HF) estimations of the Polish population based on more than 20,000 registered stem cell donors. Sequencing-based donor human leukocyte antigen (HLA) typing led to unambiguous typing results in most cases (between 94.3% for HLA-DRB1 and 96.9% for HLA-B). HF estimations were carried out with a new, validated implementation of the expectation-maximization algorithm that allowed processing of data with ambiguities. Our results confirm several earlier results, for example, the relative commonness of the haplotype A*25:01 g, B*18:01 g, C*12:03, DRB1*04:01 in the Polish population. Because of the large sample size, we were able to obtain results of unprecedented accuracy. The estimated population-specific HFs were then used to analyze questions of strategic donor registry planning. Simulated matching probabilities by donor file size suggest that there is a need for intense donor recruitment efforts in Poland despite the large German donor registry and the genetic relatedness of both populations. Based on the current German registry size of approximately 4 million donors, the recruitment of 100,000 Polish donors would produce a stronger increase in matching probabilities for Polish patients than the recruitment of 3.3 million additional German donors.