House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.

BACKGROUND: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. METHODS: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. RESULTS: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. CONCLUSIONS: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.

BACKGROUND: A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. OBJECTIVE: We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. METHODS: A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. RESULTS: The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. CONCLUSION: An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.

MOTIVATION: The numbers of finished and ongoing genome projects are increasing at a rapid rate, and providing the catalog of genes for these new genomes is a key challenge. Obtaining a set of well-characterized genes is a basic requirement in the initial steps of any genome annotation process. An accurate set of genes is needed in order to learn about species-specific properties, to train gene-finding programs, and to validate automatic predictions. Unfortunately, many new genome projects lack comprehensive experimental data to derive a reliable initial set of genes. RESULTS: In this study, we report a computational method, CEGMA (Core Eukaryotic Genes Mapping Approach), for building a highly reliable set of gene annotations in the absence of experimental data. We define a set of conserved protein families that occur in a wide range of eukaryotes, and present a mapping procedure that accurately identifies their exon-intron structures in a novel genomic sequence. CEGMA includes the use of profile-hidden Markov models to ensure the reliability of the gene structures. Our procedure allows one to build an initial set of reliable gene annotations in potentially any eukaryotic genome, even those in draft stages. AVAILABILITY: Software and data sets are available online at http://korflab.ucdavis.edu/Datasets.

The emergence of next-generation sequencing platforms led to resurgence of research in whole-genome shotgun assembly algorithms and software. DNA sequencing data from the Roche 454, Illumina/Solexa, and ABI SOLiD platforms typically present shorter read lengths, higher coverage, and different error profiles compared with Sanger sequencing data. Since 2005, several assembly software packages have been created or revised specifically for de novo assembly of next-generation sequencing data. This review summarizes and compares the published descriptions of packages named SSAKE, SHARCGS, VCAKE, Newbler, Celera Assembler, Euler, Velvet, ABySS, AllPaths, and SOAPdenovo. More generally, it compares the two standard methods known as the de Bruijn graph approach and the overlap/layout/consensus approach to assembly.

Euroglyphus maynei is a house dust mite commonly found in homes worldwide and is the source of allergens that sensitize and induce allergic reactions in humans. It is the source of species-specific allergens as well as allergens that are cross-reactive with the allergens from house dust mites Dermatophagoides farinae and D. pteronyssinus, and the ectoparasitic scabies mite Sarcoptes scabiei. The genomics, proteomics and molecular biology of E. maynei and its allergens have not been as extensively investigated as those of D. farinae, D. pteronyssinus, and S. scabiei where natural and recombinant allergens from these species have been characterized. Until now, little was known about the genome of E. maynei and it allergens but this information will be important for producing recombinant allergens for diagnostic and therapeutic purposes and for understanding the allergic response mechanism by immune effector cells that mediate the allergic reaction. We sequenced and assembled the 59 Mb E. maynei genome to aid the identification of homologs for known allergenic proteins. The predicted proteome shared orthologs with D. farinae and S. scabiei, and included proteins with homology to more than 30 different groups of allergens. However, the majority of allergen candidates could not be assigned as clear orthologs to known mite allergens. The genomic sequence data, predicted proteome, and allergen homologs identified from E. maynei provide insight into the relationships among astigmatid mites and their allergens, which should allow for the development of improved diagnostics and immunotherapy.

Dermatophagoides pteronyssinus is the European dust mite and a major source of human allergens. Here, we present the first draft genome sequence of the mite, as well as the ab initio gene prediction and functional analyses that will facilitate comparative genomic analyses with other mite species.

BACKGROUND: The disease scabies, caused by the ectoparasitic mite, Sarcoptes scabiei, causes significant morbidity in humans and other mammals worldwide. However, there is limited data available regarding the molecular basis of host specificity and host-parasite interactions. Therefore, we sought to produce a draft genome for S. scabiei and use this to identify molecular markers that will be useful for phylogenetic population studies and to identify candidate protein-coding genes that are critical to the unique biology of the parasite. METHODS: S. scabiei var. canis DNA was isolated from living mites and sequenced to ultra-deep coverage using paired-end technology. Sequence reads were assembled into gapped contigs using de Bruijn graph based algorithms. The assembled genome was examined for repetitive elements and gene annotation was performed using ab initio, and homology-based methods. RESULTS: The draft genome assembly was about 56.2 Mb and included a mitochondrial genome contig. The predicted proteome contained 10,644 proteins, ~67 % of which appear to have clear orthologs in other species. The genome also contained more than 140,000 simple sequence repeat loci that may be useful for population-level studies. The mitochondrial genome contained 13 protein coding loci and 20 transfer RNAs. Hundreds of candidate salivary gland protein genes were identified by comparing the scabies mite predicted proteome with sialoproteins and transcripts identified in ticks and other hematophagous arthropods. These include serpins, ferritins, reprolysins, apyrases and new members of the macrophage migration inhibitory factor (MIF) gene family. Numerous other genes coding for salivary proteins, metabolic enzymes, structural proteins, proteins that are potentially immune modulating, and vaccine candidates were identified. The genes encoding cysteine and serine protease paralogs as well as mu-type glutathione S-transferases are represented by gene clusters. S. scabiei possessed homologs for most of the 33 dust mite allergens. CONCLUSION: The draft genome is useful for advancing our understanding of the host-parasite interaction, the biology of the mite and its phylogenetic relationship to other Acari. The identification of antigen-producing genes, candidate immune modulating proteins and pathways, and genes responsible for acaricide resistance offers opportunities for developing new methods for diagnosing, treating and preventing this disease.

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either Pacific Biosciences (PacBio) or Oxford Nanopore technologies and achieves a contig NG50 of >21 Mbp on both human and Drosophila melanogaster PacBio data sets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

While a number of allergens from house dust mites have been described, much remains to be discovered about allergens from storage mites. Here, next-generation sequencing was combined with MS/MS shotgun proteomics to identify proteins, especially potential allergens from Tyrophagus putrescentiae, commonly found in stored food products, especially flour. cDNAs of suspected allergens were cloned and expressed from bacterial cells, and recombinant allergens were tested for binding to IgE in sera from T. putrescentiae-sensitive patients. These analyses identified three previously uncharacterized allergens, Tyr p 28, Tyr p 35, and Tyr p 36, which have been officially assigned by the WHO/IUIS Allergen Nomenclature Sub-committee. Recombinant proteins rTyr p 28, rTyr p 35, and rTyr p 36 bound with 47.1%, 82.4%, and 70.6% of T. putrescentiae-sensitive sera. We provide here a new method to identify allergens by the combination of transcriptomic and proteomic approaches.

BACKGROUND: House dust mites (HDMs) such as Dermatophagoides farinae and D. pteronyssinus represent major causes of perennial allergy. HDM proteomes are currently poorly characterized, with information mostly restricted to allergens. As of today, 33 distinct allergen groups have been identified for these 2 mite species, with groups 1 and 2 established as major allergens. Given the multiplicity of IgE-reactive mite proteins, potential additional allergens have likely been overlooked. OBJECTIVE: To perform a comprehensive characterization of the transcriptomes, proteomes and allergomes of D. farinae and D. pteronyssinus in order to identify novel allergens. METHODS: Transcriptomes were analyzed by RNA sequencing and de novo assembly. Comprehensive mass spectrometry-based analyses proteomes were combined with two-dimensional IgE reactivity profiling. RESULTS: Transcripts from D. farinae and D. pteronyssinus were assembled, translated into protein sequences and used to populate derived sequence databases in order to inform immunoproteomic analyses. A total of 527 and 157 proteins were identified by bottom-up MS analyses in aqueous extracts from purified HDM bodies and fecal pellets, respectively. Based on high sequence similarities (>71% identity), we also identified 2 partial and 11 complete putative sequences of currently undisclosed D. pteronyssinus counterparts of D. farinae registered allergens. Immunoprofiling on 2D-gels revealed the presence of unknown 23 kDa IgE reactive proteins in both species. Following expression of non-glycosylated recombinant forms of these molecules, we confirm that these new allergens react with serum IgEs from 42% (8/19) of HDM-allergic individuals. CONCLUSIONS: Using combined transcriptome and immunoproteome approaches, we provide a comprehensive characterization of D. farinae and D. pteronyssinus allergomes. We expanded the known allergen repertoire for D. pteronyssinus and identified two novel HDM allergens, now officially referred by the International Union of Immunological Societies (IUIS) Nomenclature Subcommittee as Der f 36 and Der p 36.

Aquaporins (AQPs), or water channel proteins, are highly conserved across species. These transmembrane proteins promote water and solute transport across cell membranes. No AQPrelated proteins have been identified in mites to date. The present study used transcriptomics (RNAsequencing) to identify potential AQPs in the mite species Blomia tropicalis. Molecular cloning techniques were then used to obtain the fulllength gene sequences encoding these AQP family members, and bioinformatics analyses were used to categorize them based on similarity to AQPs in other species. This approach led to the identification of 5 putative AQPcoding sequences, known as BlotAQP15 (GenBank accession numbers: KX655540, KX655541, KX655542, KX655543 and KX655544, respectively), which were indexed into all three subgroups, i.e., AQPs, aquaglyceroporins and superAQPs. To the best of our knowledge, these represent the first known AQPs in any mite species. Further studies are required to investigate their functional roles in water transport and their potential as drug targets.

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.

The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1-3, 6, 7, 10, 11, 13-18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens.

Dermatophagoides farinae mite is a predominant source of indoor allergens causing high incidence of allergy worldwide. People with different genetic background respond differently to the mite components, and thus the component-resolved diagnosis (CRD) is preferred to the conventional allergy test based on crude mite extract. In this study, proteome and culprit components in the D. farinae whole body extract that sensitized the allergic patients were studied by using SDS-PAGE (1DE) and 2DE-IgE immunoblotting followed by LC-MS/MS and database search for protein identification. From the 1DE, the mite extract revealed 105 proteins that could be classified into seven functionally different groups: allergens, structural components, enzymes, enzyme inhibitor, receptor proteins, transporters, and binding/regulatory/cell signaling proteins. From the 2DE, the mite extract produced 94 spots; 63 were bound by IgE in sera of 20 D. farinae allergic patients. One more protein that was not revealed by the 2DE and protein staining reacted with IgE in 2 allergic patients. Proteins in 40 spots could be identified as 35 different types. Three of them reacted to IgE of >50% of the allergic patients, and hence they are major allergens: tropomyosin or Der f 10 (75%), aconitate hydratase (70%), and one uncharacterized protein (55%). Aconitate hydratase is a novel D. farinae major allergen unraveled in this study. Several mite minor allergens that have never been previously reported are also identified. The data have clinical applications in the component-resolved diagnosis for tailor-designed allergen-specific immunotherapy.

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 x 10(2) to 1.4 x 10(3) per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of

'Candidatus Cardinium' is an intracellular endosymbiont or parasite frequently occurring in invertebrates including mites and ticks. In this work we report Cardinium bacteria in Astigmata mites and explore their incidence in synanthropic species. Amplification of a 776 bp bacterial 16S rRNA gene fragment, using specific primers, enabled identification of closely related Cardinium sequences in 13 laboratory-reared populations of mites. In addition, Cardinium sequences were identified in three wild mite populations. Large scale screening of these populations showed 100% prevalence of Cardinium, representing the highest incidence compared to other major Chelicerate groups.

Many insects harbor inherited bacterial endosymbionts. Although some of them are not strictly essential and are considered facultative, they can be a key to host survival under specific environmental conditions, such as parasitoid attacks, climate changes, or insecticide pressures. The whitefly Bemisia tabaci is at the top of the list of organisms inflicting agricultural damage and outbreaks, and changes in its distribution may be associated to global warming. In this work, we have sequenced and analyzed the genome of Cardinium cBtQ1, a facultative bacterial endosymbiont of B. tabaci and propose that it belongs to a new taxonomic family, which also includes Candidatus Amoebophilus asiaticus and Cardinium cEper1, endosymbionts of amoeba and wasps, respectively. Reconstruction of their last common ancestors' gene contents revealed an initial massive gene loss from the free-living ancestor. This was followed in Cardinium by smaller losses, associated with settlement in arthropods. Some of these losses, affecting cofactor and amino acid biosynthetic encoding genes, took place in Cardinium cBtQ1 after its divergence from the Cardinium cEper1 lineage and were related to its settlement in the whitefly and its endosymbionts. Furthermore, the Cardinium cBtQ1 genome displays a large proportion of transposable elements, which have recently inactivated genes and produced chromosomal rearrangements. The genome also contains a chromosomal duplication and a multicopy plasmid, which harbors several genes putatively associated with gliding motility, as well as two other genes encoding proteins with potential insecticidal activity. As gene amplification is very rare in endosymbionts, an important function of these genes cannot be ruled out.

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI-inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.