Roche GS FLX Research Articles

An online microbial data management system for poring over the equine microbiome

The soil bacteria diversity and corresponding environmental data made available here are from a 72-field plot experiment testing the effect of pulse frequency in nine wheat-based rotation systems, in the semiarid prairie. The data include sequences of the V6–V8 regions of bacterial 16S rDNA from soil and root extracts, generated using Roche GS FLX Titanium technology, and associated environmental data, specifically levels of soil organic carbon, total carbon, total nitrogen, total phosphorus, pH, electrical conductivity, and extractible sulfate sulfur, copper, iron, manganese, zinc, potassium, nitrate nitrogen, phosphate phosphorus, calcium, and magnesium in the 0–15 cm soil layer, and mineral nitrogen and phosphate in the 0–120 cm soil layer. The grain yield of wheat in the last (4th) phase of the crop rotation systems is also given. The data can be used in meta-analyses of the effect of pea, lentil and chickpea in wheat-based cropping systems on soil bacterial diversity or for monitoring the evolution of soil bacteria communities in cultivated prairie soils in the context of climate change. Samples were collected between 2012 and 2014.

Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones

Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant's genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on β-amyrin synthase and is similar to that found in other plants. The second, subsidiary (“backburner”) pathway is catalyzed by camelliol C synthase and yields β-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyltransferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.

De novo annotation of the transcriptome of the Northern Wheatear (Oenanthe oenanthe).

We have sequenced a partial transcriptome of the Northern Wheatear (Oenanthe oenanthe), a species with one of the longest migrations on Earth. The transcriptome was constructed de novo using RNA-Seq sequence data from the pooled mRNA of six different tissues: brain, muscle, intestine, liver, adipose tissue and skin. The samples came from nine captive-bred wheatears collected at three different stages of the endogenous autumn migratory period: (1) lean birds prior the onset of migration, (2) during the fattening stage and (3) individuals at their migratory body mass plateau, when they have almost doubled their lean body mass. The sample structure used to build up the transcriptome of the Northern Wheatears concerning tissue composition and time guarantees the future survey of the regulatory genes involved in the development of the migratory phenotype. Through the pre-migratory period, birds accomplish outstanding physical and behavioural changes that involve all organ systems. Nevertheless, the molecular mechanisms through which birds synchronize and control hyperphagia, fattening, restlessness increase, immunity boosting and tuning the muscles for such endurance flight are still largely unknown. The use of RNA-Seq has emerged as a powerful tool to analyse complex traits on a broad scale, and we believe it can help to characterize the migratory phenotype of wheatears at an unprecedented level. The primary challenge to conduct quantitative transcriptomic studies in non-model species is the availability of a reference transcriptome, which we have constructed and described in this paper. The cDNA was sequenced by pyrosequencing using the Genome Sequencer Roche GS FLX System; with single paired-end reads of about 400 bp. We estimate the total number of genes at 15,640, of which 67% could be annotated using Turkey and Zebra Finch genomes, or protein sequence information from SwissProt and NCBI databases. With our study, we have made a first step towards understanding the migratory phenotype regarding gene expression of a species that has become a model to study birds long-distance migrations.

Exploring the heat-responsive chaperones and microsatellite markers associated with terminal heat stress tolerance in developing wheat.

Global warming is a major threat for agriculture and food security, and in many cases the negative impacts are already apparent. Wheat is one of the most important staple food crops and is highly sensitive to the heat stress (HS) during reproductive and grain-filling stages. Here, whole transcriptome analysis of thermotolerant wheat cv. HD2985 was carried out at the post-anthesis stage under control (22±3°C) and HS-treated (42°C, 2h) conditions using Illumina Hiseq and Roche GS-FLX 454 platforms. We assembled ~24 million (control) and ~23 million (HS-treated) high-quality trimmed reads using different assemblers with optimal parameters. De novo assembly yielded 52,567 (control) and 59,658 (HS-treated) unigenes. We observed 785 transcripts to be upregulated and 431 transcripts to be downregulated under HS; 78 transcripts showed >10-fold upregulation such as HSPs, metabolic pathway-related genes, etc. Maximum number of upregulated genes was observed to be associated with processes such as HS-response, protein-folding, oxidation-reduction and photosynthesis. We identified 2008 and 2483 simple sequence repeats (SSRs) markers from control and HS-treated samples; 243 SSRs were observed to be overlying on stress-associated genes. Polymorphic study validated four SSRs to be heat-responsive in nature. Expression analysis of identified differentially expressed transcripts (DETs) showed very high fold increase in the expression of catalytic chaperones (HSP26, HSP17, and Rca) in contrasting wheat cvs. HD2985 and HD2329 under HS. We observed positive correlation between RNA-seq and qRT-PCR expression data. The present study culminated in greater understanding of the heat-response of tolerant genotype and has provided good candidate genes for the marker development and screening of wheat germplasm for thermotolerance.

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes.

Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform ( Palmer et al., 2014 ). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

De Novo Analysis of the Transcriptome of Meloidogyne enterolobii to Uncover Potential Target Genes for Biological Control.

Meloidogyne enterolobii is one of the obligate biotrophic root-knot nematodes that has the ability to reproduce on many economically-important crops. We carried out de novo sequencing of the transcriptome of M. enterolobii using Roche GS FLX and obtained 408,663 good quality reads that were assembled into 8193 contigs and 31,860 singletons. We compared the transcripts in different nematodes that were potential targets for biological control. These included the transcripts that putatively coded for CAZymes, kinases, neuropeptide genes and secretory proteins and those that were involved in the RNAi pathway and immune signaling. Typically, 75 non-membrane secretory proteins with signal peptides secreted from esophageal gland cells were identified as putative effectors, three of which were preliminarily examined using a PVX (pGR107)-based high-throughput transient plant expression system in Nicotiana benthamiana (N. benthamiana). Results showed that these candidate proteins suppressed the programmed cell death (PCD) triggered by the pro-apoptosis protein BAX, and one protein also caused necrosis, suggesting that they might suppress plant immune responses to promote pathogenicity. In conclusion, the current study provides comprehensive insight into the transcriptome of M. enterolobii for the first time and lays a foundation for further investigation and biological control strategies.

De novo transcriptome sequencing of a non-model polychaete species

Transcriptome sequencing is a useful method for studying gene-sequences data, especially in non-model organisms whose genomic sequences are yet to be determined. Indeed, even without any genome reference, transcripts can be assembled de novo to produce a genome-scale transcription map. Here we describe a de novo transcriptome assembly for the polychaete worm Ophryotrocha diadema, generated from 454-sequencing (Roche GS-FLX Titanium Sequencing). We describe the sequencing, the assembly and functional annotation of EST sequences, and the level of transcriptome coverage provided by our sequence data. The sequences we assembled represent a wide depiction of expressed genes from throughout the lifespan of O. diadema. We found gene sequences to be part of a broad range of functions (i.e. biological and metabolic processes, immune system, reproductive processes, development and growth, signalling and response to stimuli) and we therefore built the first transcriptomic reference for Ophryotrocha polychaete worms. These results encourage us to focus our investigation on many aspects of O. diadema and to extend our research to co-generic species.

The complete mitochondrial genome of Crassostrea gasar (Bivalvia: Ostreidae)

The complete mitochondrial genome of Crassostrea gasar was sequenced using the Ion Proton technology in combination with 454 Roche GS-FLX plataform data. We assembled a 17,686 bp complete circular mitochondrial genome, containing 13 protein-coding genes, a major non-coding region (MNR), two ribosomal RNA genes and 24 transfer RNA genes. Phylogenetic analysis of concatenated amino acid sequences from mitochondria showed monophyletic clades formed with high bootstrap values. This is the first complete mitochondrial sequence of an oyster from South America. Mitogenome sequence was deposited in GenBank under the accession number KR856227.

Analysis of the Transcriptome of the Infective Stage of the Beet Cyst Nematode, H. schachtii.

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii.

Harnessing Next Generation Sequencing in Climate Change: RNA-Seq Analysis of Heat Stress-Responsive Genes in Wheat (Triticum aestivum L.).

Wheat is a staple food worldwide and provides 40% of the calories in the diet. Climate change and global warming pose a threat to wheat production, however, and demand a deeper understanding of how heat stress might impact wheat production and wheat biology. However, it is difficult to identify novel heat stress associated genes when the genomic information is not available. Wheat has a very large and complex genome that is about 37 times the size of the rice genome. The present study sequenced the whole transcriptome of the wheat cv. HD2329 at the flowering stage, under control (22°±3°C) and heat stress (42°C, 2 h) conditions using Illumina HiSeq and Roche GS-FLX 454 platforms. We assembled more than 26.3 and 25.6 million high-quality reads from the control and HS-treated tissues transcriptome sequences respectively. About 76,556 (control) and 54,033 (HS-treated) contigs were assembled and annotated de novo using different assemblers and a total of 21,529 unigenes were obtained. Gene expression profile showed significant differential expression of 1525 transcripts under heat stress, of which 27 transcripts showed very high (>10) fold upregulation. Cellular processes such as metabolic processes, protein phosphorylation, oxidations-reductions, among others were highly influenced by heat stress. In summary, these observations significantly enrich the transcript dataset of wheat available on public domain and show a de novo approach to discover the heat-responsive transcripts of wheat, which can accelerate the progress of wheat stress-genomics as well as the course of wheat breeding programs in the era of climate change.

Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

BackgroundBenzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts.ResultsIn order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal (www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities.ConclusionsThis study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0596-0) contains supplementary material, which is available to authorized users.

Microbial assemblages on a cold-water coral mound at the SE Rockall Bank (NE Atlantic): interactions with hydrography and topography

Abstract. This study characterizes the microbial community composition over Haas Mound, one of the most prominent cold-water coral mounds of the Logachev Mound province (Rockall Bank, NE Atlantic). We outline patterns of distribution vertically – from the seafloor to the water column – and laterally – across the mound – and couple these to mound topography and hydrography. Samples of water, sediment and Lophelia pertusa were collected in 2012 and 2013 from locations that were chosen based on high definition video surveys. Temperature and current measurements were obtained at two sites at the summit and foot of Haas Mound to study near-bed hydrodynamic conditions. Overlaying water was collected from depths of 400 m as well as 5 and 10 m above the bottom using a CTD/Rosette system. Near-bottom water, sediment and L. pertusa mucus and skeleton samples were obtained with a box corer. Of all these biotopes, Roche GS-FLX amplicon sequencing targeting both Bacteria and Archaea was carried out, augmenting our understanding of deep sea microbial consortia. The pattern of similarities between samples, visualized by multi-dimensional scaling (MDS), indicates a strong link between the distribution of microbes and the specific biotopes. The microbial operational taxonomic unit (OTU) diversity was the highest in near-bottom water, which was sampled in the coral framework. For the first time, Thaumarchaeota marine group I (MGI) were found in L. pertusa mucus; Endozoicomonas was detected in skeleton, mucus and near-bottom water, whereas Mycoplasma was only detected in skeleton and near-bottom water, however not in mucus. Analysis of similarities (ANOSIM) indicates that overlaying water is well-mixed at 400 m depth but less so at 5 and 10 m above the bottom, where the composition of microbial communities differed significantly between summit, slope and off-mound. At all locations, the near-bottom water differed significantly from water at 5 m above the bottom, illustrating that the near-bottom water in between the coral framework represents a separate microbial habitat. Furthermore, the observed spatial heterogeneity in microbial communities is discussed in relation to environmental conditions.

The Transcriptome Sequence of Dientamoeba fragilis Offers New Biological Insights on its Metabolism, Kinome, Degradome and Potential Mechanisms of Pathogenicity

Dientamoeba fragilis is a human bowel parasite with a worldwide distribution. Dientamoeba was once described as a rare and harmless commensal though recent reports suggest it is common and potentially pathogenic. Molecular data on Dientamoeba is scarce which limits our understanding of this parasite. To address this, sequencing of the Dientamoeba transcriptome was performed. Messenger RNA was extracted from cultured Dientamoeba trophozoites originating from clinical stool specimens, and sequenced using Roche GS FLX and Illumina HiSeq technologies. In total 6,595 Dientamoeba transcripts were identified. These sequences were analysed using the BLAST2GO software suite and via BLAST comparisons to sequences available from TrichDB, GenBank, MEROPS and kinase.com. Several novel KEGG pathway maps were generated and gene ontology analysis was also performed. These results are thoroughly discussed guided by knowledge available for other related protozoa. Attention is paid to the novel biological insights afforded by this data including peptidases and kinases of Dientamoeba, as well as its metabolism, novel chemotherapeutics and possible mechanisms of pathogenicity. Currently, this work represents the largest contribution to our understanding of Dientamoeba molecular biology and also represents a major contribution to our understanding of the trichomonads generally, many of which are important pathogens of humans and animals.

Comparison of the genome profiles between head and body lice

The body louse (Pediculus humanus humanus) is known to have diverged from the head louse (P. humanus capitis) but genomic differences between these two subspecies still remain unexplored. To compare genomic profiles between head and body lice, whole genome sequences of head lice were determined by next generation sequencing methods based on both Illumina Genome analyzer and Roche GS FLX pyrosequencing and compared with the reference genome sequences of the body louse. Total consensuses generated by mapping to the body louse genome in conjunction with de novo assembly of head louse genome sequences revealed a head louse genome size of 110Mbp with a 96% coverage of the body louse genome sequences. A total of 12,651 genes were predicted from the head louse genome sequences although more precise assembly and functional annotation of the genome is required for a more accurate gene count. Among the 873 genes that were putatively specific to the head louse, 15 genes were confirmed to be transcribed in both head and body lice, suggesting the previously estimated gene number of the body louse was likely underestimated. The single nucleotide polymorphism analysis showed that the nucleotide diversity of genome between head and body lice was 2.2%, which was larger than that of the transcriptome between head and body lice. An endosymbiont genome analysis showed that the composition of endosymbionts in head lice was similar to that of body lice and Candidatus Riesia pediculicola was the primary endosymbiont in both head and body lice.

Transcriptome analysis of gill tissue of Atlantic cod Gadus morhua L. from the Baltic Sea

The Atlantic cod (Gadus morhua L.) is one of the most ecologically and economically important marine fish species in the North Atlantic Ocean. Using Roche GS-FLX 454 pyrosequencing technique 962,516 reads, representing 379Mbp of the Baltic cod transcriptome, were obtained. Data was assembled into 14,029 contigs of which 100% displayed homology to the Atlantic cod transcriptome. Despite a high similarity between transcripts, evidence for significant differences between Baltic and Atlantic cod was found.

Gene expression profiling of laser microdissected airway smooth muscle tissue in asthma and atopy

Asthma and atopy share common characteristics including type 2 helper-T-cell-mediated inflammation. However, only asthma is associated with variable airways obstruction. The complex cellular and molecular pathways distinguishing asthma and atopy can now be captured by transcriptomic analysis (RNA-Seq). We hypothesized that the transcriptomic profile of airway smooth muscle (ASM) distinguishes atopic asthma from atopic healthy controls. First, we compared the ASM transcriptomic profiles of endobronchial biopsies between glucocorticoid-free, atopic asthma patients, and atopic and nonatopic healthy controls. Second, we investigated the association between ASM transcriptomic profiles and airway function. Twelve asthma patients and 12 control subjects (six atopic, six nonatopic) underwent bronchoscopy. RNA of laser-dissected ASM from 96 bronchial biopsy specimens was sequenced with Roche GS FLX. Gene networks were identified using Ingenuity Pathway Analysis. RNA-Seq reads were assumed to follow a negative binomial distribution. With the current sample size, the estimated false discovery rate was approximately 1%. One hundred and seventy four ASM genes were differentially expressed between asthma patients and atopic controls, 108 between asthma patients and nonatopic controls, and 135 between atopic and nonatopic controls. A set of eight genes discriminated asthma patients from nonasthmatic controls, irrespective of atopy. Four of these genes (RPTOR, VANGL1, FAM129A, LEPREL1) were associated with airway hyper-responsiveness (P<0.05). Airway smooth muscle from asthma patients can be distinguished from that of atopic and nonatopic control subjects by a specific gene expression profile, which is associated with airway hyper-responsiveness.

"Deep" sequencing accuracy and reproducibility using Roche/454 technology for inferring co-receptor usage in HIV-1.

Next generation, “deep”, sequencing has increasing applications both clinically and in disparate fields of research. This study investigates the accuracy and reproducibility of “deep” sequencing as applied to co-receptor prediction using the V3 loop of Human Immunodeficiency Virus-1. Despite increasing use in HIV co-receptor prediction, the accuracy and reproducibility of deep sequencing technology, and the factors which can affect it, have received only a limited level of investigation. To accomplish this, repeated deep sequencing results were generated using the Roche GS-FLX (454) from a number of sources including a non-homogeneous clinical sample (N = 47 replicates over 18 deep sequencing runs), and a large clinical cohort from the MOTIVATE and A400129 studies (N = 1521). For repeated measurements of a non-homogeneous clinical sample, increasing input copy number both decreased variance in the measured proportion of non-R5 using virus (p<<0.001 and 0.02 for single replicates and triplicates respectively) and increased measured viral diversity (p<0.001; multiple measures). Detection of sequences with a mean abundance less than 1% abundance showed a 2 fold increase in median coefficient of variation (CV) in repeated measurements of a non-homogeneous clinical sample, and a 2.7 fold increase in CV in the MOTIVATE/A400129 dataset compared to sequences with ≥1% abundance. An unexpected source of error included read position, with low accuracy reads occurring more frequently towards the edge of sequencing regions (p<<0.001). Overall, the primary source of variability was sampling error caused by low input copy number/minority species prevalence, though other sources of error including sequence intrinsic, temporal, and read-position related errors were detected.

High-throughput analysis and characterization of Astragalus membranaceus transcriptome using 454 GS FLX.

Astragalus membranaceus (Fisch.) Bge (AR), one of the most important medicinal plants in Asia, was found to exhibit various bioactivities. Due to limited genomic and transcriptomic data, the biosynthetic pathway of the major bioactive compound in AR, is currently unclear. In this study, 454 GS FLX technology was employed to produce a substantial expressed sequence tag (EST) dataset from the AR. In all, 742721 high-quality reads from the AR were produced using Roche GS FLX Titanium. A total of 9893 unique sequences were obtained and annotated by a similarity search against the public databases, and involved in the secondary metabolic pathway, which would facilitate deciphering the molecular mechanism of secondary metabolism in AR. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. GO revealed the unique sequences that could be assigned to 34 vocabularies. In the KEGG mapping, unique sequences were established as associated with 46 biochemical pathways. These results provided the largest EST collections in AR and will contribute to biosynthetic and biochemical studies that lead to drug improvement. With respect to the genes related to metabolism and biosynthesis pathway were also found. Our work demonstrated the utility of 454 GS FLX as a method for the rapid and cost-effective identification of AR transcriptome, and this EST dataset will be a powerful resource for further studies such as taxonomy, molecular breeding, and secondary metabolism in AR.

Sequencing and analysis of the transcriptome of the acorn worm Ptychodera flava, an indirect developing hemichordate

Hemichordates are the sister group of echinoderms, and together they are closely related to chordates within the deuterostome lineage. Therefore, hemichordates represent an important animal group for the understanding of both the evolution of developmental mechanisms in deuterostome animals and the origin of chordates. Recently, the majority of studies investigating hemichordates have focused on the direct-developing enteropneust hemichordate Saccoglossus kowalevskii; few have focused on the indirect-developing hemichordates, partly because of the lack of extensive genomic resources in these animals. In this study, we report the sequencing and analysis of a transcriptome from an indirect-developing enteropneust hemichordate Ptychodera flava. We sequenced a mixed cDNA library from six developmental stages using the Roche GS FLX Titanium System to generate more than 879,000 reads. These reads were assembled into 17,990 contigs with an average length of 1316bp. We found that 60% of the assembled contigs, along with 28% of the unassembled singleton reads, had significant hits to sequences in the NCBI database by a BLASTx search, and we also annotated these sequences and obtained Gene Ontology (GO) terms for 6744 contigs and 5802 singletons. We further identified candidate P. flava transcripts corresponding to genes involved in major developmental signaling pathways, including the Wnt, Notch and TGF-β signaling pathways. Using available genome/transcriptome datasets from the direct-developing hemichordate S. kowalevskii, the echinoderm Strongylocentrotus purpuratus and the chordate Branchiostoma floridae, we found that 90%, 80% and 73% of the annotated protein sequences in these respective species matched our P. flava transcriptome in a homology search. We also constructed a database for the P. flava transcriptome, and researchers can easily access this dataset online. Our dataset significantly increases the amount of available P. flava sequence data and can serve as a reference transcriptome for future studies using this species.

Near-complete genome sequencing of swine vesicular disease virus using the Roche GS FLX sequencing platform.

Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation.