DR2S: an integrated algorithm providing reference-grade haplotype sequences from heterozygous samples.

BACKGROUND: High resolution HLA genotyping of donors and recipients is a crucially important prerequisite for haematopoetic stem-cell transplantation and relies heavily on the quality and completeness of immunogenetic reference sequence databases of allelic variation. RESULTS: Here, we report on DR2S, an R package that leverages the strengths of two sequencing technologies-the accuracy of next-generation sequencing with the read length of third-generation sequencing technologies like PacBio's SMRT sequencing or ONT's nanopore sequencing-to reconstruct fully-phased high-quality full-length haplotype sequences. Although optimised for HLA and KIR genes, DR2S is applicable to all loci with known reference sequences provided that full-length sequencing data is available for analysis. In addition, DR2S integrates supporting tools for easy visualisation and quality control of the reconstructed haplotype to ensure suitability for submission to public allele databases. CONCLUSIONS: DR2S is a largely automated workflow designed to create high-quality fully-phased reference allele sequences for highly polymorphic gene regions such as HLA or KIR. It has been used by biologists to successfully characterise and submit more than 500 HLA alleles and more than 500 KIR alleles to the IPD-IMGT/HLA and IPD-KIR databases.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 1).

Currently, stem cell donor registries include more than 35 million potential donors worldwide to provide HLA-matched stem cell products for patients in need of an unrelated donor transplant. DKMS is a leading stem cell donor registry with more than 9 million donors from Germany, Poland, the United States, the United Kingdom, India and Chile. DKMS donors have donated hematopoietic stem cells more than 80,000 times. Many aspects of donor registry work are closely related to topics from immunogenetics or population genetics. In this two-part review article, we describe, analyse and discuss these areas of donor registry work by using the example of DKMS. Part 1 of the review gives a general overview on DKMS and includes typical donor registry activities with special focus on the HLA system: high-throughput HLA typing of potential stem cell donors, HLA haplotype frequencies and resulting matching probabilities, and donor file optimization with regard to HLA diversity.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 2).

DKMS is a leading stem cell donor registry with more than 9 million donors. Donor registry activities share many touch points with topics from immunogenetics or population genetics. In this two-part review article, we deal with these aspects of donor registry work by using the example of DKMS. In the second part of the review, we focus on donor typing of non-HLA genes, the impact of donor age, gender and CMV serostatus on donation probabilities, the identification of novel HLA, KIR and MIC alleles by high-throughput donor typing, the activities of the Collaborative Biobank and pharmacogenetics in the donor registry context.

Bioinformatics Strategies, Challenges, and Opportunities for Next Generation Sequencing-Based HLA Genotyping.

The advent of next generation sequencing (NGS) has altered the face of genotyping the human leukocyte antigen (HLA) system in clinical, stem cell donor registry, and research contexts. NGS has led to a dramatically increased sequencing throughput at high accuracy, while being more time and cost efficient than precursor technologies. This has led to a broader and deeper profiling of the key genes in the human immunogenetic make-up. The rapid evolution of sequencing technologies is evidenced by the development of varied short-read sequencing platforms with differing read lengths and sequencing capacities to long-read sequencing platforms capable of profiling full genes without fragmentation. Concomitantly, there has been development of a diverse set of computational analyses and software tools developed to deal with the various strengths and limitations of the sequencing data generated by the different sequencing platforms. This review surveys the different modalities involved in generating NGS HLA profiling sequence data. It systematically describes various computational approaches that have been developed to achieve HLA genotyping to different degrees of resolution. At each stage, this review enumerates the drawbacks and advantages of each of the platforms and analysis approaches, thus providing a comprehensive picture of the current state of HLA genotyping technologies.

Genetic basis of allochronic differentiation in the fall armyworm.

BACKGROUND: Very little is known on how changes in circadian rhythms evolve. The noctuid moth Spodoptera frugiperda (Lepidoptera: Noctuidae) consists of two strains that exhibit allochronic differentiation in their mating time, which acts as a premating isolation barrier between the strains. We investigated the genetic basis of the strain-specific timing differences to identify the molecular mechanisms of differentiation in circadian rhythms. RESULTS: Through QTL analyses we identified one major Quantitative trait chromosome (QTC) underlying differentiation in circadian timing of mating activity. Using RADtags, we identified this QTC to be homologous to Bombyx mori C27, on which the clock gene vrille is located, which thus became the major candidate gene. In S. frugiperda, vrille showed strain-specific polymorphisms. Also, vrille expression differed significantly between the strains, with the rice-strain showing higher expression levels than the corn-strain. In addition, RT-qPCR experiments with the other main clock genes showed that pdp1, antagonist of vrille in the modulatory feedback loop of the circadian clock, showed higher expression levels in the rice-strain than in the corn-strain. CONCLUSIONS: Together, our results indicate that the allochronic differentiation in the two strains of S. frugiperda is associated with differential transcription of vrille or a cis-acting gene close to vrille, which contributes to the evolution of prezygotic isolation in S. frugiperda.

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.

Expression of the toxin-antitoxin genes yefM(Lrh), yoeB(Lrh) in human Lactobacillus rhamnosus isolates.

Lactobacilli are important microorganisms in various activities, for example, diary products, meat ripening, bread and pickles, but, moreover, are associated directly with human skin and cavities (e.g., mouth, gut, or vagina). Some of them are used as probiotics. Therefore, the molecular biological investigation of these bacteria is important. Earlier we described several toxin antitoxin systems (type II) in lactobacilli. Here, we describe the structure and transcriptional regulation of genes, encoding TA system YefM-YoeB(Lrh) in three strains of Lactobacillus rhamnosus comparing stationary and exponential growth phases, the influence of stress factors and mRNA stability. The same TA system is responding to physiological and stress conditions differently in related strains. Using primer extension and RLM-RACE methods we determined three transcription start sites of RNAs in the operon. The promoter region of the operon is preceded by a conserved BOX element occurring at multiple positions in the genomes of L. rhamnosus strains. Downstream of and partially overlapping with the 3' end of the yoeB(Lrh) toxin gene, a divergently transcribed unexpected RNA was detected.

Within-population variability in a moth sex pheromone blend: genetic basis and behavioural consequences.

Evolutionary diversification of sexual communication systems in moths is perplexing because signal and response are under stabilizing selection in many species, and this is expected to constrain evolutionary change. In the moth Heliothis virescens, we consistently found high phenotypic variability in the female sex pheromone blend within each of four geographically distant populations. Here, we assess the heritability, genetic basis and behavioural consequences of this variation. Artificial selection with field-collected moths dramatically increased the relative amount of the saturated compound 16:Ald and decreased its unsaturated counterpart Z11-16:Ald, the major sex pheromone component (high line). In a cross between the high- and low-selected lines, one quantitative trait locus (QTL) explained 11-21% of the phenotypic variance in the 16:Ald/Z11-16:Ald ratio. Because changes in activity of desaturase enzymes could affect this ratio, we measured their expression levels in pheromone glands and mapped desaturase genes onto our linkage map. A delta-11-desaturase had lower expression in females producing less Z11-16:Ald; however, this gene mapped to a different chromosome than the QTL. A model in which the QTL is a trans-acting repressor of delta-11 desaturase expression explains many features of the data. Selection favouring heterozygotes which produce more unsaturated components could maintain a polymorphism at this locus.

Chlamydia psittaci: new insights into genomic diversity, clinical pathology, host-pathogen interaction and anti-bacterial immunity.

The distinctive and unique features of the avian and mammalian zoonotic pathogen Chlamydia (C.) psittaci include the fulminant course of clinical disease, the remarkably wide host range and the high proportion of latent infections that are not leading to overt disease. Current knowledge on associated diseases is rather poor, even in comparison to other chlamydial agents. In the present paper, we explain and summarize the major findings of a national research network that focused on the elucidation of host-pathogen interactions in vitro and in animal models of C. psittaci infection, with the objective of improving our understanding of genomics, pathology, pathophysiology, molecular pathogenesis and immunology, and conceiving new approaches to therapy. We discuss new findings on comparative genome analysis, the complexity of pathophysiological interactions and systemic consequences, local immune response, the role of the complement system and antigen presentation pathways in the general context of state-of-the-art knowledge on chlamydial infections in humans and animals and single out relevant research topics to fill remaining knowledge gaps on this important yet somewhat neglected pathogen.

Submitting Novel Full-Length HLA, MIC, and KIR Alleles with TypeLoader2.

The Immuno Polymorphism Database (IPD) plays a pivotal role for immunogenetics. Due to technical limitations, genotyping often focuses on specific key regions like the antigen recognition domain (ARD) for HLA genotyping, and the databases are populated accordingly. More recently, though, modern next generation sequencing (NGS) assays allow using larger gene segments or even complete genes for genotyping. It is therefore essential that the databases are updated with complete genetic reference sequences to fully serve current and future applications. However, the process of manually annotating and submitting full-length allele sequences to IPD is time-consuming and error-prone, which may discourage HLA-genotyping laboratories or researchers from submitting full-length sequences of novel alleles.Here, we detail the process of preparing and submitting novel HLA, MIC, and KIR alleles to ENA and IPD using TypeLoader2, a convenient software tool developed to streamline this process by automating the sequence annotation, the creation of all necessary files, as well as parts of the submission process itself. The software is freely available from GitHub ( https://github.com/DKMS-LSL/typeloader ).

Full-Length Characterization of Novel HLA-DRB1 Alleles for Reference Database Submission.

The prerequisite for successful HLA genotyping is the integrity of the large allele reference database IPD-IMGT/HLA. Consequently, it is in the laboratories' best interest that the data quality of submitted novel sequences is high. However, due to its long and variable length, the gene HLA-DRB1 presents the biggest challenge and as of today only 16% of the HLA-DRB1 alleles in the database are characterized in full length. To improve this situation, we developed a protocol for long-range PCR amplification of targeted HLA-DRB1 alleles. By subsequently combining both long-read and short-read sequencing technologies, our protocol ensures phased and error-corrected sequences of reference grade quality. This dual redundant reference sequencing (DR2S) approach is of particular importance for correctly resolving the challenging repeat regions of DRB1 intron 1. Until today, we used this protocol to characterize and submit 384 full-length HLA-DRB1 sequences to IPD-IMGT/HLA.

The plasmidome of a Salmonella enterica serovar Derby isolated from pork meat.

The complete nucleotide sequences of four plasmids hosted by a Salmonella enterica serovar. Derby strain 6MK1 isolated from pork were determined by shotgun Sanger sequencing. A 107,637 base pairs (bp) conjugative plasmid pSD107 containing 150 putative coding sequences (CDS) could be assigned to the narrow host range incompatibility group IncI1. A detailed annotation of all CDS was carried out, revealing the presence of genes needed for plasmid replication, conjugal transfer, plasmid partitioning and stability as well as resistance to antimicrobials. The resistance determinants dhfrA1, aadA1, qacEΔ1, sul1 (supplied by a class 1 integron), blaTEM-1b (carried by a truncated Tn2 flanked by IS26), sul2 and strAB confer multidrug resistance to the host bacterium. In addition to pSD107, three small cryptic plasmids pSD4.0, pSD4.6 and pSD5.6 were identified, showing significant sequence similarities to already known replicons of Escherichia coli and S. enterica. In conjugation experiments performed on solid medium, pSD107 was successfully transferred to a nalidixic acid resistant E. coli DH5α, mobilizing pSD4.0 and, more infrequently, also pSD4.6. All transferred plasmids were stably propagated in the recipient strain without selective pressure for approximately 66 generations. The absolute plasmid copy numbers were determined in real time PCR experiments, revealing an approximate 1:1:1:1 ratio of the four replicons compared to the chromosome. The evolutionary position of pSD107 within the IncI1 family of plasmids was inferred from a maximum likelihood phylogenetic tree and by comparison of genetic key elements in a set of 17 IncI1 reference plasmids.

High population frequencies of MICA copy number variations originate from independent recombination events.

MICA is a stress-induced ligand of the NKG2D receptor that stimulates NK and T cell responses and was identified as a key determinant of anti-tumor immunity. The MICA gene is located inside the MHC complex and is in strong linkage disequilibrium with HLA-B. While an HLA-B*48-linked MICA deletion-haplotype was previously described in Asian populations, little is known about other MICA copy number variations. Here, we report the genotyping of more than two million individuals revealing high frequencies of MICA duplications (1%) and MICA deletions (0.4%). Their prevalence differs between ethnic groups and can rise to 2.8% (Croatia) and 9.2% (Mexico), respectively. Targeted sequencing of more than 70 samples indicates that these copy number variations originate from independent nonallelic homologous recombination events between segmental duplications upstream of MICA and MICB. Overall, our data warrant further investigation of disease associations and consideration of MICA copy number data in oncological study protocols.

Complete genome sequences of four mammalian isolates of Chlamydophila psittaci.

Chlamydia psittaci is an obligate intracellular zoonotic pathogen primarily of birds, but it is also known to infect a variety of mammalian species. Here we report the genomes of four strains isolated from sheep (C19/98), pigs (01DC11), cattle (02DC15), and humans (08DC60).

Full-length de novo sequence of the Chlamydophila psittaci type strain, 6BC.

Chlamydophila psittaci is an obligate intracellular zoonotic pathogen, mainly of birds. It is the causative agent of psittacosis in birds and humans. Here we report the full-length de novo genome sequence of the avian isolate 6BC, the type strain of the species C. psittaci.

Allochronic separation versus mate choice: nonrandom patterns of mating between fall armyworm host strains.

Assortative mating may result from intrinsic individual mating preferences or from assortment traits not requiring expression of preferences. Assortment traits are phenotypes expressed in both sexes that enhance the probability of encountering individuals possessing similar trait values. In the noctuid moth Spodoptera frugiperda, it has been suggested that nonrandom mating between two host strains is caused by a temporal assortment trait-that is, differential timing of calling and copulation during the night. By experimental manipulation of this trait in controlled mate-choice experiments, we investigated whether mating by same-strain individuals is enhanced mainly by the allochronic shift of mating activity or is also affected by time-independent intrinsic mating preferences. The observed patterns suggest that nonrandom mating between the two host strains in the laboratory is shaped by an interaction of both effects that is dominated by mating preferences during the first encounter night. This interaction changes over time as the preferences become weaker on subsequent nights. Males were less restricted than females with regard to both the time shift in mating activity and mate preferences. Although the nature of the mate-preference mechanism remains elusive, its restriction to females suggests that male-produced close-range pheromones emitted during courtship play a role.

Paralogous mitochondrial control region in the giant tiger shrimp, Penaeus monodon (F.) affects population genetics inference: A cautionary tale.

The mitochondrial control region (mtCR) is a widely used genetic marker for phylogenetic, phylogeographic and population genetic inference. The analysis of mtCR in 115 Indonesian specimens of the giant tiger shrimp, Penaeus monodon, revealed 26 individuals yielding a second - apparently paralogous - sequence in addition to the putatively authentic mitochondrial haplotype. The paralogous haplotypes fell into two major haplogroups that are highly diverged with respect to the authentic mitochondrial haplotypes (average pairwise sequence divergence of 12.5% and 5.0%, respectively). A comparison with published mtCR sequences of P. monodon showed that the paralogous contaminant sequences were inadvertently included in a series of recent population genetic studies, leading to seriously compromised conclusions about genetic diversity and differentiation. The prevalence of the paralogous haplotypes throughout the sampled Indo-Pacific populations is highly skewed: From African and Indian individuals only paralogs have been sequenced, while they are completely absent from Australian individuals. This suggests that geographically unequally distributed allelic variants at binding sites of the primer pair ordinarily used to amplify mtCR in P. monodon suppressed the amplification of authentic mtCR in a wide range of samples.

HLA-E diversity unfolded: Identification and characterization of 170 novel HLA-E alleles.

HLA-E is a member of the nonclassical HLA class Ib genes. Even though it is structurally highly similar to the classical HLA class Ia genes, it is less diverse and only 45 alleles and 12 proteins were known in December 2019 (IPD-IMGT/HLA, release 3.38.0). Since 2017, we have genotyped over 3 million voluntary stem cell donors for HLA-E by sequencing the most relevant allele-determining bases of exons 2 and 3. As expected, most donors harbor the two predominant alleles HLA-E*01:01 and/or HLA-E*01:03. However, in 1666 (0.05%) of our samples we detected 345 distinct novel HLA-E sequences. The most frequent one was identified in 162 samples and has by now been named HLA-E*01:114. To characterize these novel alleles in full-length, we used both short-read Illumina and long-read PacBio sequencing to obtain fully phased and highly accurate sequences. This resulted in 234 submissions to IPD-IMGT/HLA comprising 170 novel HLA-E alleles, which encode for 93 novel HLA-E proteins, as well as 64 confirmations or sequence extensions. Consequently, the number of HLA-E alleles in the database (release 3.42.0) has now increased to 256 HLA-E alleles and 110 HLA-E proteins.

Time-shifted reproductive behaviours among fall armyworm (Noctuidae: Spodoptera frugiperda) host strains: evidence for differing modes of inheritance.

The noctuid moth Spodoptera frugiperda consists of two strains associated with different larval host plants (most notably corn and rice). These strains exhibit differential temporal patterns of female calling and copulation during scotophase, with the corn strain more active earlier in the night. We investigated strain-specific constraints in reproductive timing, mating interactions between the two strains, and the mode of inheritance of timing of female calling, male calling, copulation and oviposition. We observed an allochronic shift of all reproductive behaviours by approximately 3 h and a parallel shift of nonreproductive locomotor activity, suggesting involvement of the circadian clock. The corn strain was more variable in the timing of calling and copulation than the rice strain. Rice strain females were more restricted in the timing of copulation than rice strain males, while such differences between the sexes were not apparent in the corn strain. There were significant interactions between the strains affecting onset times of copulation and male calling. The four investigated reproductive traits differed in their modes of inheritance: timing of female and male calling exhibited strong maternal effects, timing of copulation was controlled by a combination of maternal effects and corn strain dominant autosomal factors, and timing of oviposition was inherited in a corn strain dominant fashion. We conclude that the allochronic separation of reproduction between fall armyworm strains is asymmetric, less pronounced than previously thought, and under complex genetic control.