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Aim Next-generation sequencing (NGS) provides a cost and time efficient approach for routine HLA typing. Analysis software typically presumes a diploid genotype with counts of sequencing reads from homozygous or heterozygous genotypes. However, chimeric samples with more than two alleles per locus and imbalanced read counts may occur, for instance, after partially HLA-mismatched hematopoietic stem cell transplantation. Detection of alleles at even very low allele frequencies can then become diagnostically relevant in the context of post-transplant relapse. Here, we describe and validate a novel algorithm for rare HLA allele detection using NGS data. Methods Sequencing reads are mapped against expected HLA-alleles in a chimeric DNA-sample. Only polymorphic nucleotide positions differentiating the expected allele sequences are considered for further analysis. Reads are classified based on the frequency of discriminatory di-nucleotides. The classification algorithm was validated using in vitro admixtures of DNA samples down to allelic frequencies of 0.5 percent based on NGS data targeting exons 2 and 3 for the HLA genes A, B, C, DQB1, DRB1, and DPB1. Results We found that sensitivity and specificity are allele and locus-specific and depend on the number of sequence differences between the alleles. In each of the analyzed admixture samples a subset of the 12 amplicons harbored sufficient sequence differences to detect genotypes at dilutions as low as 0.5 percent. Fig. 1 shows an example for 3 amplicons. Conclusions The proposed algorithm may support the application of cost effective NGS for sensitive detection of HLA microchimerism in different settings including transplantation and pregnancy. Download : Download high-res image (200KB) Download : Download full-size image

Molecular-level Monte Carlo simulations are performed to validate equation of state approaches for the description of the N2/CH4/C2H6 vapour–liquid equilibria under conditions relevant to Titan’s lower atmosphere. The Monte Carlo simulations confirm the validity of the equation of state approaches, so that both provide a reliable description of the unknown composition of cloud droplets in this region of Titan’s atmosphere. Furthermore, the models are compared with experimental data from laboratory studies of aerosol droplets that contain N2, CH4 and C2H6. Good agreement is also found here.

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many "bioorthogonal" reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3-7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN-tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli , and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.

Previous microarray analyses revealed that in Bradyrhizobium japonicum , about 100 genes are induced by genistein, an isoflavonoid secreted by soybean. This includes the three genes freC , freA , and freB (systematic designations bll4319 , bll4320 , and bll4321 ), which are likely to form a genistein-, daidzein-, and coumestrol-inducible operon and to encode a multidrug efflux system. Upstream of freCAB and in the opposite orientation, FrrA (systematic designation Blr4322), which has similarity to TetR-type regulators, is encoded. A deletion of frrA leads to increased expression of freB in the absence of an inducer. We identified the correct translational start codon of frrA and showed that the gene is inducible by genistein and daidzein. The protein, which was heterologously expressed and purified from Escherichia coli , binds to two palindrome-like DNA elements (operator A and operator B), which are located in the intergenic region between freC and frrA . The replacement of several nucleotides or the insertion of additional spacer nucleotides prevented binding. Binding of FrrA was also affected by the addition of genistein. By mapping the transcription start sites, we found that operator A covers the transcriptional start site of freC and operator B is probably located between the −35 regions of the two divergently oriented genes. Operator A seems to be conserved in a few similar gene constellations in other proteobacteria. Our data indicate that in B. japonicum , besides NodD1 (the LysR family) and NodVW (a two-component response regulator), a third regulator type (a TetR family member) which responds to the plant signal molecules genistein and daidzein exists.

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis . These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.

The investigation and modelling of permafrost distribution, particularly in areas of discontinuous permafrost, is challenging due to spatial heterogeneity, remoteness of measurement sites and data scarcity. We have designed a strategy for standardizing different local data sets containing evidence of the presence or absence of permafrost into an inventory for the entire European Alps. With this brief communication, we present the structure and contents of this inventory. This collection of permafrost evidence not only highlights existing data and allows new analyses based on larger data sets, but also provides complementary information for an improved interpretation of monitoring results.

Spectroscopic studies of the homogeneous and heterogeneous crystallization of supercooled ethane aerosol droplets performed under conditions representative of those in Titan's lower atmosphere are presented. Pure ethane aerosols and both internally- and externally-mixed ensembles of ethane/acetylene and ethane/carbon dioxide aerosols were generated in a bath gas cooling cell and their freezing dynamics monitored using infrared absorption spectroscopy. A detailed overview of the spectroscopic signatures of pure ethane and mixed ethane/acetylene and ethane/carbon dioxide aerosols and their phase-dependence is provided. The ice-nucleating efficiencies of acetylene and carbon dioxide aerosols were compared, as were the efficiencies of freezing via an immersion or contact freezing mechanism. The spectral data provided will be of significant use for remote sensing applications, while the nucleation studies have important consequences for models of Titan's ethane clouds.

Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-L-lysine (7) and δ-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.

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.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

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.

Aim KIR2DL1 is a member of the killer-cell immunoglobulin-like receptor (KIR) family, which is an important factor of the human immune system. KIR genes are key regulators of natural killer cell activity and partly bind to proteins of the human leukocyte antigen (HLA) family, e.g. HLA-B and -C in the case of KIR2DL1. Likely due to the complexity of the KIR locus with its extensive genetic variation, only little is known about the impact of allelic variation. Here, we identified novel KIR2DL1 alleles by routine high-throughput exon-based KIR genotyping and subsequently created full-length reference sequences. We analysed coding and non-coding variation to identify possible systematic differences between alleles. Methods We sequenced whole 16 kb amplicons of KIR2DL1 using shotgun sequencing (Illumina MiSeq) and single molecule real time (SMRT) sequencing (PacBio Sequel). Using the R package DR2S, we combined phase information from SMRT sequencing with the accuracy of shotgun sequencing to generate phased full-length sequences. Results We successfully generated 60 distinct KIR2DL1 allele sequences from 45 specifically selected samples. This includes 41 novel alleles and 17 distinct alleles that were previously only partially characterised. The sequence analysis revealed three deep allelic groups separated by 164 variable positions. Allelic variants of two groups harbour mutually exclusive nucleotides, while a third group of alleles may harbour nucleotides of both the other groups. The three groups are mostly separated by non-coding SNPs, indels, and a T homopolymer of fixed length in one and variable length in two groups. Some separating variants exist in most exons and alter the amino acid sequence of the leader peptide, the extracellular D1- and D2-domains containing the HLA binding sites as well as the cytoplasmatic domain. Conclusions Our sequencing efforts resulted in a 4-fold increase in known full-length sequences of KIR2DL1, enabling further research on this KIR gene. We gained insights into systematic differences at the sequence level which might be responsible for or indicative of medically relevant allelic differences. Especially variation in the D2-domain has been shown to be involved in binding to HLA proteins and may as such be clinically relevant.

The toxin colicin E3 targets the 30S subunit of bacterial ribosomes and cleaves a phosphodiester bond in the decoding center. We present the crystal structure of the 70S ribosome in complex with the cytotoxic domain of colicin E3 (E3-rRNase). The structure reveals how the rRNase domain of colicin binds to the A site of the decoding center in the 70S ribosome and cleaves the 16S ribosomal RNA (rRNA) between A1493 and G1494. The cleavage mechanism involves the concerted action of conserved residues Glu62 and His58 of the cytotoxic domain of colicin E3. These residues activate the 16S rRNA for 2' OH-induced hydrolysis. Conformational changes observed for E3-rRNase, 16S rRNA and helix 69 of 23S rRNA suggest that a dynamic binding platform is required for colicin E3 binding and function.

Strong evidence for ethane clouds in various regions of Titan's atmosphere has recently been found. Ethane is usually assumed to exist as ice particles in these clouds, although the possible role of liquid and supercooled liquid ethane droplets has been recognized. Here, we report on infrared spectroscopic measurements of ethane aerosols performed in the laboratory under conditions mimicking Titan's lower atmosphere. The results clearly show that liquid ethane droplets are significantly stabilized by methane gas which is ubiquitous in Titan's nitrogen atmosphere — a phenomenon that does not have a counterpart for water droplets in Earth's atmosphere. Our data imply that supercooled ethane droplets are much more abundant in Titan's clouds than previously anticipated. Possibly, these liquid droplets are even more important for cloud processes and the formation of lakes than ethane ice particles., The Astrophysical Journal Letters, 712 (1), ISSN:1967-2014, ISSN:2041-8213

All in a knot. The smallest riboswitch forms a pseudoknot in solution. This is demonstrated for preQ(1) class I aptamers by mutational analysis in combination with (1)H NMR-based structure probing. How pseudoknot formation mediates the mRNA response through its expression platform is now open for investigation.

This protocol describes an efficient method for the preparation of riboswitch domains comprising up to approximately 200 nt containing site-specific nucleoside modifications. The strategy is based on enzymatic ligation of chemically synthesized RNA fragments. The design of ligation sites strictly follows the criterion that all fragments comprise less than approximately 50 nt. This allows the researcher to rely on custom synthesis services and to utilize the large pool of commercially available, functionalized nucleoside phosphoramidites for solid-phase RNA synthesis. Importantly, this design renders utmost flexibility to position a chemical modification (e.g., a fluorescence label) within the RNA. Selection of the appropriate ligation type (using T4 RNA or T4 DNA ligase) is subordinate to the criteria above and is detailed in the protocol. The whole concept is demonstrated for 2-aminopurine containing thiamine pyrophosphate responsive riboswitch domains that are applied in fluorescence spectroscopic folding studies. Labeled samples in 5-35 nmol quantities are obtained within 3-4 d, not including the time for fragment synthesis.

In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.

By using a structure-based fluorescence spectroscopic approach, we have examined the folding of an adenine-responsive riboswitch that regulates translation initiation. We observed adaptive recognition of the ligand for the aptamer domain of adenosine deaminase (add) mRNA from Vibrio vulnificus, and revealed pronounced conformational changes even in the preorganized loop-loop region that is distant from the binding site. Importantly, the full-length riboswitch domain, which has a potential translational repressor stem is able to form a binary complex with adenine, and does not act as a folding trap to inhibit binding. The aptamer that is extended by the expression platform therefore remains fully responsive to its ligand; this is in contrast to the previously investigated pbuE A-riboswitch, which becomes trapped in a nonresponsive terminator fold. Consequently, the latter must employ complex response mechanisms, such as operating in kinetic-control mode or using transcriptional pausing, to provide time for the aptamer portion to fold and to bind. The different behavior of the riboswitches can be rationalized by their distinct sequence interface between the aptamer and expression platform. For the add A-riboswitch, our data suggest a thermodynamically driven response mechanism.

The metabotropic receptor P2Y1 is necessary for full ADP-induced platelet activation and is localized on various intrinsic renal cells, including mesangial cells, podocytes, and endothelial cells. To date, nothing is known about the role of the P2Y1 receptor during inflammatory renal disease. The role of the P2Y1 receptor was investigated using 22 P2Y1 gene–deficient (−/−) and 27 wild-type (wt) mice during the time course of passive crescentic nephrotoxic glomerulonephritis. Six P2Y1 −/− and six wt mice served as undiseased controls. Renal tissues were harvested on days 1, 10, and 28 after disease induction. No renal phenotype was found in P2Y1 −/− versus wt mice. In contrast, during crescentic glomerulonephritis, approximately 50% of all wt mice died, whereas all P2Y1 −/− mice survived. Renal function as assessed by creatinine clearance measurements, glomerulosclerosis, and tubulointerstitial injury indices as well as glomerular and interstitial matrix expansion were improved significantly in P2Y1 −/− compared with wt mice. These changes were preceded by reduced glomerular and peritubular capillary rarefaction indices in P2Y1 −/− compared with wt mice. The alteration of the rates of both peritubular apoptosis and endothelial cell proliferation suggests improved capillary preservation in P2Y1 −/− mice early in disease (day 10) and an additional enhanced repair reaction in P2Y1 −/− mice at the late time point (day 28), whereas injury on day 1 seemed to be equivalent in both groups. It is concluded that loss of P2Y1 receptor function safeguards against capillary loss, fibrosis, and death by renal failure during experimental crescentic glomerulonephritis.

The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small molecule ligands exist.

We have recently introduced a basic concept for the combined chemical and enzymatic preparation of site-specifically modified 2'-methylseleno RNAs which represent useful derivatives for phasing of X-ray crystallographic data during their three-dimensional structure determination. Here, we introduce the first synthesis of an appropriate guanosine phosphoramidite, which complements the thus far established set of 2'-methylseleno-modified uridine, cytidine, and adenosine building blocks for solid-phase synthesis. The novel building block was readily incorporated into RNA. Importantly, it was the 2'-methylseleno-guanosine-labeled RNA that allowed us to reveal the reversible oxidation/reduction behavior of the Se moiety and thus it represents a valuable contribution to the understanding of the action of threo-1,4-dimercapto-2,3-butanediol (DTT) required during solid-phase synthesis, deprotection, and crystallization of selenium-containing RNA. In addition, we investigated 2'-methylseleno RNA with respect to crystallization properties. Our studies revealed that the Se modification significantly increases the range of conditions leading to crystal growth. Moreover, we determined the crystal structures of model RNA helices and showed that the Se modification can affect crystal packing interactions, thus potentially expanding the possibilities for obtaining the best crystal form.

The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2 0 -deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2 0 -hydroxyl was present. This implicates a functional or structural role of the 2 0 -hydroxyl group at A2451 for transpeptidation.

Genetic code expansion for unnatural amino acid mutagenesis has, until recently, been limited to cell culture. We demonstrate the site-specific incorporation of unnatural amino acids into proteins in Drosophila melanogaster at different developmental stages, in specific tissues and in a subset of cells within a tissue. This approach provides a foundation for probing and controlling processes in this established metazoan model organism with a new level of molecular precision.

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labeling proteins with any probe. Here we describe the genetic encoding of dienophile-bearing unnatural amino acids into proteins expressed in Escherichia coli and mammalian cells using the pyrrolysyl-tRNA synthetase/tRNACUA pair and its variants. We describe the rapid fluorogenic labeling of proteins containing these unnatural amino acids in vitro, in E. coli, and in live mammalian cells with tetrazine-fluorophore conjugates in a bioorthogonal Diels-Alder reaction with inverse electron demand. These approaches have been extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on the labeling and imaging field.

Aim The killer cell immunoglobulin-like receptor (KIR) gene family is seen to play an important role in unrelated hematopoietic stem cell transplantations. Allele level KIR genotyping may improve the donor selection process. The IPD-KIR database currently comprises 753 alleles. With our newly established high-throughput workflow for KIR allele-level typing, we discovered a vast number of previously unknown variations of KIR alleles. Methods We perform cost efficient KIR allele level genotyping based on a paired-end short-amplicon approach by NGS using Illumina HiSeq 2500 instruments. The amplicons cover exons 3, 4, 5, 7, 8 and 9 of each KIR gene. Our neXtype software was adapted to report KIR genotyping results at allelic Level. Results Starting in October 2016, we added KIR allele-level typing to the DKMS standard genotyping profile for new potential donors and already applied it to more than 500,000 samples. Since then, we have detected more than 5000 distinct novel SNP sequences, see Fig. 1, i.e. sequences that differ from the reported alleles by at least one nucleotide. About half of the novel sequences exhibit new protein sequences. In addition, we identified many alleles with so far unreported combinations of described exon sequences. We analyzed the distribution of new variants according to ethnic self-assessment of recruited donors. Conclusions The ability to genotype KIR genes at allelic level in a high-throughput framework gives the opportunity to detect a huge number of unknown sequences on a rather short time scale. Consequently, the database of currently named alleles can be extended significantly and thus gives input to studies around the complex KIR gene Family.

Aim Nanopore sequencing is one of the latest DNA sequencing technologies, in which single molecules are sequenced directly. It offers the potential of read lengths of tens of kilobases. However, the reads are relatively error prone. Especially the length of homopolymers and repetitive sequences cannot be determined accurately. Here we present the typing results for HLA samples using Oxford Nanopore Technologies (ONT). Methods We have developed NGSengine® (GenDx), a software package to perform HLA typing based on NGS data. NGSengine uses an integrated HLA typing approach which includes determining the HLA locus, aligning reads, phasing and typing into a single method. At DKMS Life Science Lab, the MinION sequencer from ONT has been used for full-length HLA sequencing. We have used a benchmarking set of 30 samples, 10 each for HLA-A, HLA-B and HLA-C to explore 3 successive Nanopore chemistries R7.3, R9 and R9.4. Results Using the successive ONT chemistries, we have mapped the template-complement (2D) reads to the known reference alleles using Burrows-Wheeler Aligner, resulting in a mappability increasing from 98 to 99.5%. Additionally, we have used NGSengine to map the reads to the allele database, increasing the mappability from 4 to 70%. The lower mappability with NGSengine is due to the noise present within the data and the thresholds set within NGSengine. For most of the validation samples the best match presented by NGSengine corresponds with the pretyping, but all have exon mismatches in homopolymers. Noteworthy, for the samples for which the best match did not match the pretype, a Null allele was called due to the incorrect length of a homopolymer. Conclusion With the current status of MinION data quality, the results look promising but cannot yet be applied for routinge HLA typing. 1D 2 basecalling with a slight change to the chemistry (R9.5) will be available soon. We will present an update on this chemistry, and the improvements concerning homopolymers.

Aim The human killer-cell immunoglobulin-like receptor (KIR) family of genes is a key regulator of natural killer cell activity. Several studies have indicated that KIR genotypes affect hematopoietic stem cell transplantation outcome and the evidence is rising that the extensive allelic diversity discovered in KIR genes may be an important component to consider. However, high-resolution characterization of KIR genotypes has so far been applied only to smaller cohorts. Therefore, we developed a KIR genotyping approach based on NGS that would be cost-effective and amendable for high-throughput application. Methods We amplified KIR exons 3, 4, 5, 7, 8 and 9, targeting one to two exons per PCR reaction but multiplexed across all KIR genes. Amplicons of 2 × 3760 samples were combined for sequencing on Illumina HiSeq 2500 instruments together with amplicons for HLA and blood groups. The reads were mapped against the described KIR alleles (IPD-KIR Release 2.6). Based on the read coverage, we estimated the genomic copy number of each sequence feature. These sequence copy calculations enabled precise allele calling and in addition empowered us to determine gene copy numbers. Results After successful validation, we applied this workflow to genotype 500,000 registry samples. Despite the focus on throughput and cost efficiency, we achieved mostly allotype (3 digit) resolution with the exception of certain allotypes differing only in the short non-targeted exon regions. In addition, certain phasing ambiguities remained. We report the observed allele diversity and the relative abundance of alleles for a mainly Caucasian cohort. Conclusions We demonstrate that high-resolution KIR genotyping is feasible using a very cost-efficient workflow. This approach enables KIR genotyping for population genetics, disease association studies and other applications requiring large cohorts. As of October 2016 we have been applying this workflow to the analysis of all volunteer samples registering with DKMS as potential donors for hematopoietic stem cell transplantation. High-resolution KIR genotyping results will thus become available to search coordinators and may soon be used as an additional selection criterion to improve transplantation outcome.

Aim In 2013, we implemented a short amplicon based Next Generation Sequencing (NGS) high-throughput workflow for HLA donor registry typing. Data analysis is performed by the in-house typing software neXtype. Currently, over 30,000 samples per week can be typed for 6 HLA loci, ABO, RhD, CCR5, and KIR with this cost-efficient workflow in our lab. As HLA-E has been reported to impact the outcome of stem cell transplantations in certain settings, we evaluated the feasibility of low-cost high-throughput HLA-E typing. On a clinically relevant antigen recognition domain (ARD) level, there are currently 9 groups of HLA-E alleles known (Fig. 1). Methods HLA-E is amplified by PCR with two primers spanning exons 2 and 3. This amplification product is subject to sequencing on Illumina MiSeq or HiSeq 2500 instruments without fragmentation. Thereby, information about the phasing between exon 2 and exon 3 is preserved, which is an important advantage of this method since it allows genotyping at ARD-level resolution. As shown in Fig. 1, alleles HLA-E∗01:01:02, 01:03:03, 01:04, 01:05, 01:07 and 01:08N can be resolved to the allele level. The remaining alleles fall into three distinguishable G-groups, namely HLA-E∗01:01:01G, 01:03:01G and 01:03:02G. Results The primer set was tested using 384 samples. All successfully sequenced samples corresponded to the G-groups HLA-E∗01:01:01G, 01:03:01G and 01:03:02G. Validation of the workflow against pre-typed samples is projected. Conclusions Sequencing HLA-E on allelic level has been show feasible in a cost efficient high throughput workflow. As the impact of HLA-E on the transplantation outcome becomes more settled, it could be considered for inclusion into the recruitment typing profile. Download high-res image (244KB) Download full-size image

Aim While short read sequencing technologies have proven highly successful for HLA genotyping, longer reads would be desirable to resolve phasing ambiguities. The emerging nanopore sequencing platform from Oxford Nanopore Technologies (ONT) presents a promising technology capable of sequencing unfragmented molecules in the order of tens of kilobases. We demonstrate that nanopore sequencing is an increasingly viable platform for HLA genotyping by addressing questions of throughput, mappability and genotyping accuracy. Methods We sequenced a benchmarking set of 30 samples with established full length sequence information, 10 each for HLA-A, HLA-B and HLA-C, on the MinION, a nanopore sequencing device from ONT, using 3 successive chemistries R7.3, R9 and R9.4. We mapped the resulting reads, both template-only (1D) and template-complement (2D), to the reference sequences using ‘bwa’ in its ‘ont’ mode, a set of parameters tailored for MinION sequencing reads. Consensus sequences were then derived from the SAM mapping data using a routine leveraging the pysam Python library. Results We observed progressive chemistries yielding increasing read numbers, from approximately 3000 reads to 8000 reads per sample. We also observed a successive increase in mappability from 90% to 98% for 1D reads, and from 97% to 99.8% for 2D reads. We compared the consensus sequences with the reference sequences, and observed reproducibility at 98% for 1D reads and 99.8% for 2D reads. We note here that the ONT basecaller systematically undercalls polynucleotide stretches resulting in gaps explaining the consensus sequence errors. We expect to report on this issue being ameliorated by a basecaller update scheduled for May 2017 specifically aimed at accurately calling homopolymer stretches. We evaluate if improvements in basecalling are achievable by training the neural network that underlies the ONT basecaller on curated data derived from HLA references sequences. Conclusions The sequencing yield and mappability of full-length HLA alleles using nanopore sequencing could render it a suitable platform for HLA genotyping. The platform’s major disadvantage, severe undercalling of polynucleotide stretches, is expected to improve with pending updates to the basecalling software.

Aim Our current understanding of sequence variation in HLA-DPB1 is largely limited to exons 2 and 3, as only few alleles have been characterized at full length. To close these gaps in the IMGT/HLA database and to better understand patterns of polymorphisms in the HLA-DPB1 gene, we selected samples from the DKMS donor pool that represent all common and well-documented (CWD) alleles and performed phased full-length sequence characterization. In addition we characterized alleles that are currently not defined as CWD but occur at moderate to high frequencies in the DKMS donor populations. Methods We performed long-range PCR targeting the whole DPB1 gene using primers flanking the UTR-regions. The 12 kb amplicons were sequenced by two strategies on different platforms: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on PacBio® RS II instruments. Phase-defined allelic consensus sequences were generated from the long-read data and polished with the high-fidelity short reads using the DR2S software (DKMS Life Science Lab). Results We successfully sequenced the full-length DPB1 sequences of 331 samples (662 haplotypes) covering 40 common, 14 well-documented, and 40 not-CWD-defined alleles. In total, 247 distinct allelic variants were found considering also variation outside of exons 2 and 3. The 12 previously known full-length DPB1 alleles were each confirmed at least once. Most novel variation was found to reside in the intronic regions of DPB1. A phylogenetic analysis of the 247 alleles confirmed the existence of two major evolutionary branches characterized by two haplotype groups downstream of exon 2 with little internal variation and no evidence of historical recombination. These haplotype groups are tightly linked to the expression marker rs9277534 in the 3’ UTR. Conclusions We present the full-length sequences of 247 unique DPB1 alleles covering all common and all well-documented (CWD) variants. We have successfully submitted these alleles to IMGT/HLA, thus providing full genomic extension of 84 known DPB1 alleles and adding 151 novel DPB1 alleles to the database. Together, these alleles represent more than 99.9% of the DPB1 alleles encountered in the DKMS donor populations.

Aim The human killer-cell immunoglobulin-like receptors (KIR) forms a complex receptor family expressed on the surface of natural killer (NK) cells. KIR genotypes have been demonstrated to affect disease predisposition and transplantation outcome. To facilitate the selection of donors based on KIR genotypes, we added KIR to the genotyping profile for newly registered potential stem cell donors in 2014. Up to now we have KIR genotyped more than 2 million samples based on our next-generation sequencing approach. This resulted in the discovery of thousands of novel alleles. For the submission of novel alleles to the IPD-KIR Database, full-length phased sequence characterization is desirable. Therefore, we initiated a project for high quality full-length sequence characterization of novel KIR3DL1 , KIR3DL2 , and KIR3DL3 alleles. Methods We established three long-range PCRs targeting the whole genes including both UTRs resulting in PCR products of 13.5 kb ( KIR3DL1 ), 17.5 kb ( KIR3DL2 ), and 16 kb ( KIR3DL3 ). Redundant sequencing strategies were applied to sequence two independent PCR products per target: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on Pacific Biosciences Sequel Systems. Using the Dual Redundant Reference Sequencing software (DR2S, DKMS Life Science Lab), phase-defined consensus sequences were generated from the SMRT data and confirmed with the short, high-fidelity reads. Applying this approach we analyzed a set of 364 samples targeting 112 distinct novel KIR alleles (41 KIR3DL1 , 37 KIR3DL2 , and 34 KIR3DL3 ). Results The generated sequencing data revealed high specificity of the assays for KIR3DL1 , KIR3DL2 and KIR3DL3 . The approach proved capable of generating fully phased sequences spanning the entire 13 to 17 kb genes. By applying two complementary technologies, rare sequencing errors could be corrected. This enabled high-quality phase-defined characterization of novel KIR alleles without the requirement for time consuming cloning steps. Conclusions We established a method for highest quality phased whole-gene KIR sequencing and applied it to characterize more than 100 novel KIR3DL1 , KIR3DL2 , and KIR3DL3 alleles. By submitting these sequences to IPD-KIR we will complement the database for frequent but so far undescribed KIR alleles.

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labeling proteins with any probe. Here we describe the genetic encoding of dienophile-bearing unnatural amino acids into proteins expressed in Escherichia coli and mammalian cells using the pyrrolysyl-tRNA synthetase/tRNACUA pair and its variants. We describe the rapid fluorogenic labeling of proteins containing these unnatural amino acids in vitro, in E. coli, and in live mammalian cells with tetrazine-fluorophore conjugates in a bioorthogonal Diels-Alder reaction with inverse electron demand. These approaches have been extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on the labeling and imaging field.

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.

O the past 15 years a great deal of progress has been made on the discovery, rediscovery, and invention of bioorthogonal reactions between functional groups that do not react with biological entities under physiological conditions but selectively react with each other. Strategies for labeling different classes of biomolecules have been developed by coopting the biosynthetic machinery of cells to introduce molecules containing bioorthogonal functional groups. Tagging approaches have allowed some additional functional groups to be attached to proteins, and genetic code expansion and reprogramming have facilitated the site-specific incorporation of unnatural amino acids bearing bioorthogonal functional groups into proteins in bacteria, mammalian cells, and animals via the discovery and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs and orthogonal ribosomes. In addition, selective pressure incorporation and its derivatives have allowed the statistical labeling of proteins and proteomes with analogues of natural amino acids. The incorporation of unnatural amino acids bearing bioorthogonal functional groups and their chemoselective labeling has great potential for imaging and controlling individual proteins and labeling proteomes, but the ability of investigators to leverage these approaches for biological discovery will be crucially dependent on the properties of the chemical reactions used. The reactants in a bioorthogonal reaction should be kinetically, thermodynamically, and metabolically stable before the reaction takes place and not toxic to living systems. The reaction should yield stable covalent linkages with no or innocuous byproducts. Moreover, the two bioorthogonal moieties have to react selectively with each other under physiological conditions (ambient temperature and pressure, neutral pH, aqueous conditions), without either of them crossreacting with the plethora of chemical functionalities found in living cells. Despite the challenges of meeting these criteria, a number of reactions have been developed that show good biocompatibility and selectivity in living systems (see Figure 1). Some of these reactions are chemoselective with respect to many but not all biological functionalities and have been used to label proteins in vitro and on the cell surface, while other reactions have additionally been used for the more challenging task of labeling proteins inside cells or living animals. Most bioorthogonal reactions follow second-order kinetics, and their rates depend directly on the concentrations of both reaction partners as well as on the intrinsic second-order rate constant k2 [M −1 s−1] of the reaction. Rapid reactions with high second-order rate constants are therefore advantageous for labeling during biological processes that occur on a very short time scale or for the labeling of low abundance proteins. Lower abundance proteins can sometimes be labeled with a large excess of labeling reagent, but this strategy may be practically limited by solubility, off target reactions and toxicity. Bioorthogonal reactions for which one partner can be installed into proteins are summarized in Figure 1. Their second-order rate constants span 9 orders of magnitude with the fastest bioorthogonal labeling reactions reaching rates up to 10 M−1 s−1, which approaches the rate constants for many enzymatic labeling approaches. Here we briefly introduce the bioorthogonal chemistries used for labeling proteins and comment on their utility for protein labeling before providing a perspective on future directions. Amongst the first functionalities to be explored as bioorthogonal reporters were ketones and aldehydes. Under acidic conditions (pH 4−6) their carbonyl groups react with strong α-effect nucleophiles such as hydrazines and alkoxyamines. Ketone/aldehyde condensations show rather slow kinetics with second-order rate constants in the range of 10−4 to 10−3 M−1 s−1, necessitating high concentrations of labeling reagent in order to achieve good labeling, which might be problematic in terms of toxicity and background signal. In general ketone/aldehyde condensations are best suited for in vitro or cell-surface labeling, because the reaction requires an acidic pH, which is difficult to obtain inside most cellular compartments. Furthermore, inside living cells, α-effect nucleophiles may undergo side-reactions with carbonyl-bearing metabolites. A functionality that is essentially absent from biological systems and truly orthogonal in its reactivity to the majority of biological functionalities is the azide group. Azide-bearing unnatural amino acids have been incorporated into proteins and used in a variety of chemical reactions. One potential limitation of the use of azides for protein labeling is that some unnatural amino acids bearing azides appear to be reduced in some proteins examined. Azide-modified proteins have been reacted with phosphines in Staudinger ligations. This reaction has been used to label biomolecules in living cells and animals. The Staudinger ligation, however, has slow kinetics: the reaction proceeds with second-order rate constants in the low 10−3 M−1 s−1 range. In addition many of the phosphine reagents are oxidized by air or metabolic enzymes. Azides can also react with terminal alkynes in [3 + 2] cycloadditions, catalyzed by Cu salts. The CuAAC (Cucatalyzed alkyne−azide cycloaddition) reaction proceeds considerably faster than the Staudinger ligation in physiological settings. However, its reliance on the Cu catalyst is not without problems, since Cu may be toxic to living systems, and decreasing the copper concentration is generally accompanied by a large decrease in reaction rate. The development of tailored water-soluble Cu ligands and/or

Spectroscopic studies of two phase transitions of solid n-butane aerosol droplets performed under conditions representative of those in the lower atmosphere of Titan are presented. Pure n-butane aerosols and mixed ensembles of n-butane/acetylene, n-butane/carbon dioxide and n-butane/water aerosols were generated in a bath gas cooling cell at 78 K and their phase transition dynamics monitored using infrared extinction spectroscopy. For pure n-butane aerosols the volume and surface nucleation constants were found to range from JV = 10(12) -10(14) cm(-3) s(-1) and JS = 10(13) -10(15) cm(-2) s(-1), respectively, for the first observed transition, and JV = 10(9) -10(11) cm(-3) s(-1) and JS = 10(11) -10(13) cm(-2) s(-1) for the second observed transition. The phases of the n-butane aerosols were determined by comparing their spectroscopic signatures with spectra collected from thin films of liquid and solid n-butane. The first observed transition was from an amorphous-annealed phase into the metastable crystalline phase II of n-butane. The second transition was from the metastable crystalline phase II into the crystalline phase III. The effect of the presence of a second aerosol substance (acetylene, carbon dioxide or water) was examined; while this accelerated the first phase transition, it did not directly influence the rate of the second phase transition. The kinetic studies might be important for the understanding of cloud formation on Titan, while the spectral data provided, which include the first reported spectrum of liquid n-butane close to the melting point, are expected to be of use for remote sensing applications.

Clinical use of 2-deoxystreptamine aminoglycoside antibiotics, which target the bacterial ribosome, is compromised by adverse effects related to limited drug selectivity. Here we present a series of 4′,6′-O-acetal and 4′-O-ether modifications on glucopyranosyl ring I of aminoglycosides. Chemical modifications were guided by measuring interactions between the compounds synthesized and ribosomes harbouring single point mutations in the drug-binding site, resulting in aminoglycosides that interact poorly with the drug-binding pocket of eukaryotic mitochondrial or cytosolic ribosomes. Yet, these compounds largely retain their inhibitory activity for bacterial ribosomes and show antibacterial activity. Our data indicate that 4′-O-substituted aminoglycosides possess increased selectivity towards bacterial ribosomes and little activity for any of the human drug-binding pockets., Aminoglycoside antibiotics target the ribosome but their limited selectivity for the bacterial ribosome can cause side effects in humans. Here, the authors synthesize 4′-O-ether or 4′,6′-O-acetal modifications and show that these compounds possess increased selectivity against bacterial ribosomes.

KIR genes are recognized to play an important role for stem cell transplantation. The high polymorphism of these genes makes it difficult to achieve high resolution typing results.Based on paired-end short amplicon next generation sequencing (NGS) data from the Ilumina Miseq and HiSeq systems, neXtype was adapted to type KIR on allelic level. neXtype reports KIR allele typing results in the genotype list (GL) string format and implicitly provides gene copy numbers.neXtype copes with KIR typing challenges in several consecutive steps:In a first step based on the IPD-KIR library, each read is assigned the best matching known exon sequence using the tree-concept of neXtype. In a second step, the copy numbers of the identified allelic variants per exon are determined for a given primer batch. This estimated exon copy number is subsequently required for the scoring system. As a third step, combination of exon-specific results determines KIR alleles present in the sample. The number of valid exon combinations per gene yields the gene copy number. Currently, neXtype uses exons 3, 4, 5, 7, 8, and 9. The assignment of reads to a specific gene is a priori not possible as some alleles of different genes share identical exon DNA sequences. Therefore, valid combinations for those gene-bridging exons must be analyzed simultaneously. neXtype uses a scoring system based on the difference between the copy number as given by valid exon combinations from step three and the estimated exon copy number from step two. The exon combination with the lowest score is taken as the typing result and presented as a GL string.With this algorithm, samples with pre-known typing results were confirmed. In summary, we show that cost efficient high-throughput NGS data can be used for allele level KIR typing. As a consequence, allele level KIR typing could be included into the standard typing profile of newly registered stem cell donors, thus allowing for refined donor selection algorithms.

Aim Given the complex nature of the human HLA system, generating reliable, full-length phase-defined reference sequences for novel HLA alleles remains a substantial challenge. Here we present a computational workflow that tackles this challenge by combining the respective strengths of two types of sequencing approaches: The high read quality of Illumina short read data and the long read length of single-molecule sequencing approaches such as PacBio and Oxford Nanopore. Methods Long reads from heterozygous samples are mapped initially against a global locus-specific reference. Based on this mapping, a SNP matrix is constructed and long reads are clustered into two read sets representing the two haplotypes. These read sets are used to construct preliminary haplotype consensus sequences, against which short reads are mapped. Integrating the alignments derived from the two complementary sequencing approaches eliminates each method’s systematic biases in the final consensus sequences and generates HLA haplotypes that identify both single-nucleotide and structural variants in reference quality. Results The DR2S approach was evaluated with 90 heterozygous HLA class I samples. Error rates decreased with increasing long-read coverage. The quality gain due to augmenting long-read data with short-read data was most pronounced at low to moderate long-read coverage ( Conclusion The integration of single-molecule and sequencing-by-synthesis NGS data by DR2S eliminates both single-nucleotide and structural variation errors and thereby yields reference quality de novo sequences.

In 2014 Oxford Nanopores Technologies released the MinION, a portable miniaturized sequencer delivering multi-kb DNA reads based on nanopore sensing technology. Sequencing accuracy is improved by consecutive sequencing both strands of the DNA (2D reads). Being part of the MinION early access program we evaluated this new technology for whole gene HLA sequencing. As the sequencing yield exceeds the requirements for a targeted approach we developed a strategy for barcoding and demultiplexing of samples. Barcoded amplicons were generated by two subsequent long range PCRs covering the complete genes of HLA-A, -B and -C. Amplicons were pooled and library preparation was performed according to the Oxford Nanopore protocol (SQK-MAP006). Sequencing was performed on a MinION MKI with R7.3 flow cells. NGSengine from GenDX was used for sequence analysis and HLA genotyping. 96 randomly picked class I samples were pooled and sequenced. Over 170,000 reads were generated, approximately 65% of them as 2D reads. Two thirds of the 2D reads could be assigned to a unique barcode sequence. The HLA genotyping results were compared to allelic resolution genotypes obtained by full length sequencing the samples on both Illumina and PacBio instruments. The ONT typing results had a 95% concordance rate when considering 1st to 3rd field. Even when taking the fourth field in account concordance rate was above 90%. Given that the nanopore technology is rapidly evolving and the genotyping software has not yet been optimized for nanopore data, this study clearly underlines the great potential of the MinION for high resolution HLA genotyping.

Aim HLA-DPB1 mismatching between the patient and unrelated donor is known to increase the risk of acute graft-versus-host-disease (GvHD) after hematopoietic stem cell transplantation. If only HLA- DPB1 mismatched donors are available, the genotype defined by the Single Nucleotide Polymorphism (SNP) rs9277534 can be used to select mismatched donors that are well-tolerated. However, since rs9277534 resides within the 3′ untranslated region (UTR), it usually is not analyzed during DPB1 routine typing. To verify whether rs9277534 can be inferred from standard DPB1-typing, we analyzed over 25,000 samples of mostly Caucasian origin for linkage between rs9277534 and high resolution HLA-DPB1 typing results based on sequencing exons 2 and 3. Methods rs9277534 located in the 3′ UTR was amplified by PCR and sequenced on Illumina MiSeq or Hiseq instruments. SNP calling was performed with FasType (DKMS Life Science Lab). DPB1 typing based on sequencing exons 2 and 3 was performed as described before. Results 99.5% of the samples carried the expected rs9277534 allele based on typing of exons 2 and 3. Only in one sample (0.004%), the expected linkage between exon 2 and rs9277534 was broken. In the remaining 0.5% samples, the linkage could not be established because of remaining ambiguities due to missing phasing between exons 2 and 3. Phasing a subset of these samples with PacBio whole gene DPB1 sequencing confirmed the presence of alleles with the expected linkage to the identified rs9277534 variants. Further, we have established the rs9277534 linkage for 23 DPB1 alleles for which the 3′ UTR sequence is so far unknown within IMGT. Conclusions HLA-DPB1 typing of exons 2 and 3 is sufficient to infer rs9277534, a marker for DPB1 expression. This information could be used to select HLA-DPB1-mismatched donors that do not increase the risk of GvHD. E.W. Petersdorf: Grant/Research Support; Company/Organization; National Institutes of Health. Employee; Company/Organization; Frederick National Laboratory for Cancer Research, National Institutes of Health.

[1] Air-sea/land turbulent fluxes of momentum, sensible heat, water vapor, carbon dioxide, and ozone are discussed on the basis of eddy covariance measurements made aboard the NOAA R/V Ronald H. Brown during the Texas Air Quality Study (TexAQS) in August–September 2006. The TexAQS 2006 field campaign focused on air pollution meteorology associated primarily with ozone and aerosol transport in the Houston/Galveston region and the nearby coastal zone. The ship-based complement of instrumentation was used for the boundary layer measurements over water (the Gulf of Mexico and various harbors/bay areas) and “over land” (specifically, 80 km inside the Houston Ship Channel). In this study we focus on direct comparisons of TexAQS 2006 flux observations with the Coupled Ocean-Atmosphere Response Experiment (COARE) bulk flux algorithm to investigate possible coastal and urban area influences. It is found that the average neutral drag coefficient can be about an order of magnitude larger over very rough urban areas than over the sea surface. However, a similar effect was not observed for the scalar transfer; that is, the neutral Stanton and Dalton numbers do not change significantly over different footprint surfaces. Our data suggest that the TexAQS 2006 region was generally a sink for surface ozone whether over water or over land. The turbulent flux of carbon dioxide was mostly negative (uptake by the surface) for measurements over waters of the Gulf of Mexico and some bays, but the flux becomes positive (release to the air) for inland regions. Both ozone and carbon dioxide turbulent fluxes above land were larger in magnitude compared to the over water measurements.

Cloning and sequencing of a 47.1-kb chromosomal DNA region revealed the presence of a type III secretion system (T3SS) in Bradyrhizobium elkanii USDA61. The identified genes are likely to encode the transcriptional activator TtsI, core components of the secretion apparatus and secreted proteins. Several ORFs within the cluster are not conserved in other rhizobia. Nine tts box motifs, a promoter element of TtsI-regulated genes, were found; six of them upstream of annotated genes. For functional analyses, the rhcC2 and rhcJ genes were disrupted. These mutations had a cultivar-specific effect on nodulation. Vigna radiata cv. KPS1 developed nodules if infected with the mutant strains but not with the wild type. In contrast, V. radiata cv. CN36 was nodulated by all strains. Nodulation of rj(1) soybean depended on the T3SS. A comparison of the protein patterns from supernatants of the wild type and rhcJ mutant by two-dimensional gel electrophoresis revealed proteins that are secreted only in the wild-type background. These results show that B. elkanii encodes a functional T3SS that is involved in the interaction with host legumes.

Site-specific 2′-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2′-OCH3-modified RNA sequences, prior to the replacement of 2′-OCH3 groups with their 2′-SeCH3 counterparts. The method exploits the similar crystallization properties of 2′-OCH3- and 2′-SeCH3-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2′-SeCH3-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality.

Type III secretion systems (T3SS) have been identified in a number of rhizobia and were shown to affect symbiosis with legumes. The genes encoding the conserved core of the T3SS (rhc; rhizobium conserved) are highly conserved in all cases, although differences exist in the secreted proteins. In our preliminary study, the presence of rhcC2 and rhcJ in Bradyrhizobium elkanii was verified by PCR and sequencing of the fragments. In this study, we characterized the T3SS of B. elkanii USDA61 by DNA sequencing, mutagenesis, RT-PCR and extracellular protein analysis.