Genome Analyzer Platform Research Articles

Abstract 4875: HIVE Proteomics: Integrated, cloud-based RNA-Seq and proteomics analysis of prostate adenocarcinoma samples

Abstract Automated bottom-up proteomics workflows implemented with modern mass-spectrometry instrumentation can readily generate millions of peptide fragmentation spectra from cell-lines and clinically derived samples. The tandem mass-spectra promise to reveal wild-type and somatic mutations, insertions, and deletions and alternatively spliced isoforms translated to functional protein isoforms in tumor samples. However, the available reference proteomes represent a poor analysis substrate for the observation of protein evidence of genomic and transcript variation. The advent of cheap and fast RNA sequencing (RNA-Seq) provides an elegant solution to the lack of sample-specific reference proteomes. We anticipate that the falling cost of RNA-Seq will prompt an increasing number of proteomics labs to use contract next-gen sequencing (NGS) services to obtain RNA-Seq data to derive sample-specific reference proteomes. In contrast to public repositories of genomic variation, sample-specific RNA-Seq data captures transcribed rare, individual, cell-type, and sample-specific genomic variation. RNA-Seq-based transcripts also provide sample-specific information on observable proteins. Furthermore, paired RNA-Seq and proteomics data links gene expression and protein abundance, enabling the study of gene regulation linked to protein abundance dynamics. However, the analysis of multi-gigabyte paired mass-spectra and RNA-Seq datasets pose significant scientific and logistical challenges. Few proteomics labs have the personnel, archival data storage, computational resources, or informatics pipelines needed. The cloud-based genomics analysis platform High-performance Integrated Virtual Environment (HIVE) will provide turn-key integrated proteomics and RNA-Seq analyses to the wider proteomics community in a secure, trackable, sharable, and scalable computing platform. We will then use this integrated proteomics_RNA-Seq analysis pipeline to identify high-value mutations in prostate adenocarcinoma samples, thereby demonstrating the utility of the platform while also generating key data for future investigations. Citation Format: Hayley Dingerdissen, Raja Mazumder. HIVE Proteomics: Integrated, cloud-based RNA-Seq and proteomics analysis of prostate adenocarcinoma samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4875. doi:10.1158/1538-7445.AM2015-4875

Dynamic transcriptome profiles of skeletal muscle tissue across 11 developmental stages for both Tongcheng and Yorkshire pigs.

BackgroundThe growth and development of skeletal muscle directly impacts the quantity and quality of pork production. Chinese indigenous pig breeds and exotic species vary greatly in terms of muscle production and performance traits. We present transcriptome profiles of 110 skeletal muscle samples from Tongcheng (TC) and Yorkshire (YK) pigs at 11 developmental periods (30, 40, 55, 63, 70, 90, and 105 days of gestation, and 0, 1, 3, and 5 weeks of age) using digital gene expression on Solexa/Illumina’s Genome Analyzer platform to investigate the differences in prenatal and postnatal skeletal muscle between the two breeds.ResultsMuscle morphological changes indicate the importance of primary fiber formation from 30 to 40 dpc (days post coitus), and secondary fiber formation from 55 to 70 dpc. We screened 4,331 differentially expressed genes in TC and 2,259 in YK (log2 ratio >1 and probability >0.7). Cluster analysis showed different gene expression patterns between TC and YK pigs. The transcripts were annotated in terms of Gene Ontology related to muscle development. We found that the genes CXCL10, EIF2B5, PSMA6, FBXO32, and LOC100622249 played vital roles in the muscle regulatory networks in the TC breed, whereas the genes SGCD, ENG, THBD, AQP4, and BTG2 played dominant roles in the YK breed. These genes showed breed-specific and development-dependent differential expression patterns. Furthermore, 984 genes were identified in myogenesis. A heat map showed that significantly enriched pathways (FDR <0.05) had stage-specific functional regulatory mechanisms. Finally, the differentially expressed genes from our sequencing results were confirmed by real-time quantitative polymerase chain reaction.ConclusionsThis study detected many functional genes and showed differences in the molecular mechanisms of skeletal muscle development between TC and YK pigs. TC pigs showed slower muscle growth and more complicated genetic regulation than YK pigs. Many differentially expressed genes showed breed-specific expression patterns. Our data provide a better understanding of skeletal muscle developmental differences and valuable information for improving pork quality.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1580-7) contains supplementary material, which is available to authorized users.

Identification of differentially expressed genes related to aphid resistance in cucumber (Cucumis sativus L.).

Cucumber, a very important vegetable crop worldwide, is easily damaged by pests. Aphids (Aphis gossypii Glover) are among the most serious pests in cucumber production and often cause severe loss of yield and make fruit quality get worse. Identifying genes that render cucumbers resistant to aphid-induced damage and breeding aphid-resistant cucumber varieties have become the most promising control strategies. In this study, a Illumina Genome Analyzer platform was applied to monitor changes in gene expression in the whole genome of the cucumber cultivar ‘EP6392’ which is resistant to aphids. Nine DGE libraries were constructed from infected and uninfected leaves. In total, 49 differentially expressed genes related to cucumber aphid resistance were screened during the treatment period. These genes are mainly associated with signal transduction, plant-pathogen interactions, flavonoid biosynthesis, amino acid metabolism and sugar metabolism pathways. Eight of the 49 genes may be associated with aphid resistance. Finally, expression of 9 randomly selected genes was evaluated by qRT-PCR to verify the results for the tag-mapped genes. With the exception of 1-aminocyclopropane-1-carboxylate oxidase homolog 6, the expression of the chosen genes was in agreement with the results of the tag-sequencing analysis patterns.

Transcriptome sequencing and comparative analysis of diploid and autotetraploid Paulownia australis

Paulownia is a fast-growing tree native to China. It can provide high-quality timber and a green environment for humans and has been planted in many countries. Compared with diploid Paulownia australis, autotetraploid P. australis exhibits predominance in terms of yields, qualities, growth, and resistance to stress. However, the molecular and biologi- cal mechanisms of its predominant characteristics are still unclear. We performed paired-end sequencing of the transcriptomes of diploid and autotetraploidP. australis plants using the Illumina/Solexa Genome Analyzer platform and compared changes in the expression of genes after chromo- somedoubling. We obtainedatotal of48,445,468and46,437, 758 high-quality reads from diploid P. australis (PA2) and autotetraploid P. australis(PA4), respectively. After assembly, 90,881 (PA2) and 88,566 (PA4) unigenes were obtained. The unigenes from both transcriptomes were then assembled into 93,300 all-unigenes, which included 50,892 clusters and 42, 408 singletons. The all-unigene sequences were searched against public protein databases (Nr, Nt, Swiss-Prot, KOG, and KEGG) using BLASTX with a cutoff E-value of 10 −5 . Gene expression levels in the two libraries were compared, and a total of 8851 differentially expressed unigenes were identified. A large number of unigenes were found to be related to carbohydrate and energy metabolism, biosynthesis of cell wall, and stress tolerance. Many of the differentially expressed P. australis genes were attributable to chromosome doubling between the PA2 and PA4 plants. Functional analy- sis revealed variations in the mechanism of growth and stress resistance between PA2 and PA4.

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining

To construct a protein-protein interaction (PPI) network in hypertension patients with blood-stasis syndrome (BSS) by using digital gene expression (DGE) sequencing and database mining techniques. DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were filtered by comparing the expression levels between the different experimental groups. Then functional categories and enriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery (DAVID) to select those in the enrichment pathways. Interologous Interaction Database (I2D) was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identified by comparing the number of relationships among genes. Confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), gene ontology (GO) analysis was used to infer the functional annotations of the potential candidate genes for BSS. With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identified: DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2BE, H3F3B, CEBPB, SAT1 and GADD45A. Verified through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored. Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.

HelicoBase: a Helicobacter genomic resource and analysis platform.

BackgroundHelicobacter is a genus of Gram-negative bacteria, possessing a characteristic helical shape that has been associated with a wide spectrum of human diseases. Although much research has been done on Helicobacter and many genomes have been sequenced, currently there is no specialized Helicobacter genomic resource and analysis platform to facilitate analysis of these genomes. With the increasing number of Helicobacter genomes being sequenced, comparative genomic analysis on members of this species will provide further insights on their taxonomy, phylogeny, pathogenicity and other information that may contribute to better management of diseases caused by Helicobacter pathogens.DescriptionTo facilitate the ongoing research on Helicobacter, a specialized central repository and analysis platform for the Helicobacter research community is needed to host the fast-growing amount of genomic data and facilitate the analysis of these data, particularly comparative analysis. Here we present HelicoBase, a user-friendly Helicobacter resource platform with diverse functionality for the analysis of Helicobacter genomic data for the Helicobacter research communities. HelicoBase hosts a total of 13 species and 166 genome sequences of Helicobacter spp. Genome annotations such as gene/protein sequences, protein function and sub-cellular localisation are also included. Our web implementation supports diverse query types and seamless searching of annotations using an AJAX-based real-time searching system. JBrowse is also incorporated to allow rapid and seamless browsing of Helicobacter genomes and annotations. Advanced bioinformatics analysis tools consisting of standard BLAST for similarity search, VFDB BLAST for sequence similarity search against the Virulence Factor Database (VFDB), Pairwise Genome Comparison (PGC) tool for comparative genomic analysis, and a newly designed Pathogenomics Profiling Tool (PathoProT) for comparative pathogenomic analysis are also included to facilitate the analysis of Helicobacter genomic data.ConclusionsHelicoBase offers access to a range of genomic resources as well as tools for the analysis of Helicobacter genome data. HelicoBase can be accessed at http://helicobacter.um.edu.my.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-600) contains supplementary material, which is available to authorized users.

Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions.

BackgroundThe clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity.ResultsA software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed, we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018.ConclusionThese studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1142) contains supplementary material, which is available to authorized users.

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.

Presentation of large DNA molecules for analysis as nanoconfined dumbbells.

The analysis of very large DNA molecules intrinsically supports long-range, phased sequence information, but requires new approaches for their effective presentation as part of any genome analysis platform. Using a multi-pronged approach that marshaled molecular confinement, ionic environment, and DNA elastic properties-but tressed by molecular simulations-we have developed an efficient and scalable approach for presentation of large DNA molecules within nanoscale slits. Our approach relies on the formation of DNA dumbbells, where large segments of the molecules remain outside the nanoslits used to confine them. The low ionic environment, synergizing other features of our approach, enables DNA molecules to adopt a fully stretched conformation, comparable to the contour length, thereby facilitating analysis by optical microscopy. Accordingly, a molecular model is proposed to describe the conformation and dynamics of the DNA molecules within the nanoslits; a Langevin description of the polymer dynamics is adopted in which hydrodynamic effects are included through a Green's function formalism. Our simulations reveal that a delicate balance between electrostatic and hydrodynamic interactions is responsible for the observed molecular conformations. We demonstrate and further confirm that the "Odijk regime" does indeed start when the confinement dimensions size are of the same order of magnitude as the persistence length of the molecule. We also summarize current theories concerning dumbbell dynamics.

Discovery of structural alterations in solid tumor oligodendroglioma by single molecule analysis

BackgroundSolid tumors present a panoply of genomic alterations, from single base changes to the gain or loss of entire chromosomes. Although aberrations at the two extremes of this spectrum are readily defined, comprehensive discernment of the complex and disperse mutational spectrum of cancer genomes remains a significant challenge for current genome analysis platforms. In this context, high throughput, single molecule platforms like Optical Mapping offer a unique perspective.ResultsUsing measurements from large ensembles of individual DNA molecules, we have discovered genomic structural alterations in the solid tumor oligodendroglioma. Over a thousand structural variants were identified in each tumor sample, without any prior hypotheses, and often in genomic regions deemed intractable by other technologies. These findings were then validated by comprehensive comparisons to variants reported in external and internal databases, and by selected experimental corroborations. Alterations range in size from under 5 kb to hundreds of kilobases, and comprise insertions, deletions, inversions and compound events. Candidate mutations were scored at sub-genic resolution and unambiguously reveal structural details at aberrant loci.ConclusionsThe Optical Mapping system provides a rich description of the complex genomes of solid tumors, including sequence level aberrations, structural alterations and copy number variants that power generation of functional hypotheses for oligodendroglioma genetics.

GenePattern flow cytometry suite.

BackgroundTraditional flow cytometry data analysis is largely based on interactive and time consuming analysis of series two dimensional representations of up to 20 dimensional data. Recent technological advances have increased the amount of data generated by the technology and outpaced the development of data analysis approaches. While there are advanced tools available, including many R/BioConductor packages, these are only accessible programmatically and therefore out of reach for most experimentalists. GenePattern is a powerful genomic analysis platform with over 200 tools for analysis of gene expression, proteomics, and other data. A web-based interface provides easy access to these tools and allows the creation of automated analysis pipelines enabling reproducible research.ResultsIn order to bring advanced flow cytometry data analysis tools to experimentalists without programmatic skills, we developed the GenePattern Flow Cytometry Suite. It contains 34 open source GenePattern flow cytometry modules covering methods from basic processing of flow cytometry standard (i.e., FCS) files to advanced algorithms for automated identification of cell populations, normalization and quality assessment. Internally, these modules leverage from functionality developed in R/BioConductor. Using the GenePattern web-based interface, they can be connected to build analytical pipelines.ConclusionsGenePattern Flow Cytometry Suite brings advanced flow cytometry data analysis capabilities to users with minimal computer skills. Functionality previously available only to skilled bioinformaticians is now easily accessible from a web browser.

Genome-Wide Analysis of Differentially Expressed Genes Relevant to Rhizome Formation in Lotus Root (Nelumbo nucifera Gaertn).

Lotus root is a popular wetland vegetable which produces edible rhizome. At the molecular level, the regulation of rhizome formation is very complex, which has not been sufficiently addressed in research. In this study, to identify differentially expressed genes (DEGs) in lotus root, four libraries (L1 library: stolon stage, L2 library: initial swelling stage, L3 library: middle swelling stage, L4: later swelling stage) were constructed from the rhizome development stages. High-throughput tag-sequencing technique was used which is based on Solexa Genome Analyzer Platform. Approximately 5.0 million tags were sequenced, and 4542104, 4474755, 4777919, and 4750348 clean tags including 151282, 137476, 215872, and 166005 distinct tags were obtained after removal of low quality tags from each library respectively. More than 43% distinct tags were unambiguous tags mapping to the reference genes, and 40% were unambiguous tag-mapped genes. From L1, L2, L3, and L4, total 20471, 18785, 23448, and 21778 genes were annotated, after mapping their functions in existing databases. Profiling of gene expression in L1/L2, L2/L3, and L3/L4 libraries were different among most of the selected 20 DEGs. Most of the DEGs in L1/L2 libraries were relevant to fiber development and stress response, while in L2/L3 and L3/L4 libraries, major of the DEGs were involved in metabolism of energy and storage. All up-regulated transcriptional factors in four libraries and 14 important rhizome formation-related genes in four libraries were also identified. In addition, the expression of 9 genes from identified DEGs was performed by qRT-PCR method. In a summary, this study provides a comprehensive understanding of gene expression during the rhizome formation in lotus root.

Comparative genomic analysis of catfish linkage group 8 reveals two homologous chromosomes in zebrafish and other teleosts with extensive inter-chromosomal rearrangements

BackgroundComparative genomics is a powerful tool to transfer genomic information from model species to related non-model species. Channel catfish (Ictalurus punctatus) is the primary aquaculture species in the United States. Its existing genome resources such as genomic sequences generated from next generation sequencing, BAC end sequences (BES), physical maps, linkage maps, and integrated linkage and physical maps using BES-associated markers provide a platform for comparative genomic analysis between catfish and other model teleost fish species. This study aimed to gain understanding of genome organizations and similarities among catfish and several sequenced teleost genomes using linkage group 8 (LG8) as a pilot study.ResultsWith existing genome resources, 287 unique genes were identified in LG8. Comparative genome analysis indicated that most of these 287 genes on catfish LG8 are located on two homologous chromosomes of zebrafish, medaka, stickleback, and three chromosomes of green-spotted pufferfish. Large numbers of conserved syntenies were identified. Detailed analysis of the conserved syntenies in relation to chromosome level similarities revealed extensive inter-chromosomal and intra-chromosomal rearrangements during evolution. Of the 287 genes, 35 genes were found to be duplicated in the catfish genome, with the vast majority of the duplications being interchromosomal.ConclusionsComparative genome analysis is a powerful tool even in the absence of a well-assembled whole genome sequence. In spite of sequence stacking due to low resolution of the linkage and physical maps, conserved syntenies can be identified although the exact gene order and orientation are unknown at present. Through chromosome-level comparative analysis, homologous chromosomes among teleosts can be identified. Syntenic analysis should facilitate annotation of the catfish genome, which in turn, should facilitate functional inference of genes based on their orthology.

Comparative transcriptome analysis and marker development of two closely related Primrose species (Primula poissonii and Primula wilsonii)

BackgroundPrimula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China. Studies on population genetics, speciation and phylogeny of Primula have been impeded by a lack of genomic resources. In the present study, we sequenced the transcriptomes of two closely related primrose species, Primula poissonii and Primula wilsonii, using short reads on the Illumina Genome Analyzer platform.ResultsWe obtained 55,284 and 55,011 contigs with N50 values of 938 and 1,085 for P. poissonii and P. wilsonii, respectively, and 6,654 pairs of putative orthologs were identified between the two species. Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 877 of the pairs may be under positive selection (Ka/Ks > 0.5), and functional enrichment analysis revealed that significant proportions of the orthologs were in the categories DNA repair, stress resistance, which may provide some hints as to how the two closely related Primula species adapted differentially to extreme environments, such as habitats characterized by aridity, high altitude and high levels of ionizing radiation. It was possible for the first time to estimate the divergence time between the radiated species pair, P. poissonii and P. wilsonii; this was found to be approximately 0.90 ± 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Primers based on 54 pairs of orthologous SSR-containing sequences between the two Primula species were designed and verified. About half of these pairs successfully amplified for both species. Of the 959 single copy nuclear genes shared by four model plants (known as APVO genes), 111 single copy nuclear genes were verified as being present in both Primula species and exon-anchored and intron-spanned primers were designed for use.ConclusionWe characterized the transcriptomes for the two Primula species, and produced an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two Primula species not only revealed a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. Furthermore, we developed two sets of genetic markers, single copy nuclear genes and nuclear microsatellites (EST-SSR). Both these sets of markers will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.

Transcriptome analysis reveals differentially expressed genes associated with propamocarb response in cucumber (Cucumis sativus L.) fruit

High-throughput tag-sequencing (Tag-seq) from Illumina analysis, which is based on the Solexa Genome Analyzer platform, was applied to analyze the gene expression profiling of propamocarb (PM) treatment and control in cucumber fruit. Approximately 3.6 million complete clean sequence tags at PM treatment or control library were obtained with approximately 0.1 million distinct clean tag sequences. Approximately 41.79–43.15 % of the distinct clean tags were mapped unambiguously to the unigene database, and 32.54–33.46 % of the distinct clean tags were mapped to the cucumber genome database. The profiling analysis of the differentially expressed genes revealed the up-regulation of 546 genes and the down-regulation of 185 genes with PM response. Furthermore, the differentially expressed genes mainly linked to pesticide detoxication, response to stress/stimulus, transporter/signaling, and some important transcription factors. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription polymerase chain reaction (RT-PCR) using 16 genes independently verified the tag-mapped results. The present study reveals the comprehensive mechanisms of PM response in cucumber fruit.

CGAP: a new comprehensive platform for the comparative analysis of chloroplast genomes

BackgroundChloroplast is an essential organelle in plants which contains independent genome. Chloroplast genomes have been widely used for plant phylogenetic inference recently. The number of complete chloroplast genomes increases rapidly with the development of various genome sequencing projects. However, no comprehensive platform or tool has been developed for the comparative and phylogenetic analysis of chloroplast genomes. Thus, we constructed a comprehensive platform for the comparative and phylogenetic analysis of complete chloroplast genomes which was named as chloroplast genome analysis platform (CGAP).ResultsCGAP is an interactive web-based platform which was designed for the comparative analysis of complete chloroplast genomes. CGAP integrated genome collection, visualization, content comparison, phylogeny analysis and annotation functions together. CGAP implemented four web servers including creating complete and regional genome maps of high quality, comparing genome features, constructing phylogenetic trees using complete genome sequences, and annotating draft chloroplast genomes submitted by users.ConclusionsBoth CGAP and source code are available at http://www.herbbol.org:8000/chloroplast. CGAP will facilitate the collection, visualization, comparison and annotation of complete chloroplast genomes. Users can customize the comparative and phylogenetic analysis using their own unpublished chloroplast genomes.

Evolution of foot-and-mouth disease virus intra-sample sequence diversity during serial transmission in bovine hosts

RNA virus populations within samples are highly heterogeneous, containing a large number of minority sequence variants which can potentially be transmitted to other susceptible hosts. Consequently, consensus genome sequences provide an incomplete picture of the within- and between-host viral evolutionary dynamics during transmission. Foot-and-mouth disease virus (FMDV) is an RNA virus that can spread from primary sites of replication, via the systemic circulation, to found distinct sites of local infection at epithelial surfaces. Viral evolution in these different tissues occurs independently, each of them potentially providing a source of virus to seed subsequent transmission events. This study employed the Illumina Genome Analyzer platform to sequence 18 FMDV samples collected from a chain of sequentially infected cattle. These data generated snap-shots of the evolving viral population structures within different animals and tissues. Analyses of the mutation spectra revealed polymorphisms at frequencies >0.5% at between 21 and 146 sites across the genome for these samples, while 13 sites acquired mutations in excess of consensus frequency (50%). Analysis of polymorphism frequency revealed that a number of minority variants were transmitted during host-to-host infection events, while the size of the intra-host founder populations appeared to be smaller. These data indicate that viral population complexity is influenced by small intra-host bottlenecks and relatively large inter-host bottlenecks. The dynamics of minority variants are consistent with the actions of genetic drift rather than strong selection. These results provide novel insights into the evolution of FMDV that can be applied to reconstruct both intra- and inter-host transmission routes.

Identification of differentially expressed genes relevant to corm formation in Sagittaria trifolia.

Sagittaria trifolia is a good model of wetland plants to elucidate the formation of corm. However, few studies have been conducted to uncover the complexity of gene expression involved in corm formation. In this study, high-throughput tag-sequencing based on Solexa Genome Analyzer Platform was applied to monitor the changes in gene expression with three libraries of differentially expressed genes (DEGs) (C1 library: stolon stage, C2 library: initial swelling stage and C3 library: swelling stage) during corm formation in Sagittaria trifolia. Approximately 6.0 million tags were sequenced, and 5854021, 5983454, and 5761079 clean tags including 138319, 116804, and 101739 distinct tags were obtained after removal of low quality tags from each library, respectively. About 46% distinct tags were unambiguous tags mapping to the reference genes, and 33% were unambiguous tag-mapped genes. Totally, 20575, 19807, and 18438 were annotated in C1, C2, and C3 libraries, respectively, after mapping their functions in existing databases. In addition, we found that profiling of gene expression in C1/C2 and C2/C3 libraries were different among most of the selected 20 DEGs. Most DEGs in C1/C2 libraries were relevant to hormone synthesis and response; energy metabolism and stress response, while most of the genes in C2/C3 libraries were involved in carbohydrate metabolism. All up-regulated transcriptional factors and 16 important genes relevant to corm formation in three libraries were also identified. To further analyze the expression of 9 genes, from the results of tag-sequencing, qRT-PCR was applied. In summary, this study provides a comprehensive understanding of gene expression, during the formation of corm in Sagittaria trifolia.

頭足類の比較ゲノムプラットフォームの展望

頭足類は発達した眼と非常に高度な脳神経をもち，無脊椎動物の中で最も進化した生物の一つである。古くから発生学・進化学・神経生理学など様々な分野にわたるアプローチがなされており，脊椎動物と形態類似性を持つカメラ眼や神経内分泌系，閉鎖血管系は，頭足類の研究のみならず，脊椎動物の進化プロセスを研究する上で非常に重要な生物種である。しかしこれまで，頭足類ゲノムは解読されていない。この理由としては，頭足類ゲノムは全般的に2-5Gbとヒトを超えるゲノムサイズを持つことが珍しくなく，ゲノムを決定することが難しかったことがあげられる。こうした状況の中，2012年５月に様々な国における頭足類研究者およびゲノム研究者が集まり，頭足類ゲノム決定を推進するコンソーシアム（Cephalopod Sequencing Consortium：CephSeq）を立ち上げた。本稿では，比較ゲノム研究・比較トランスクリプトーム研究の最近の動向をふまえながら，頭足類ゲノムプラットフォームの今後の展開を紹介する。

The screening and ranking algorithm to detect DNA copy number variations

DNA Copy number variation (CNV) has recently gained considerable interest as a source of genetic variation that likely influences phenotypic differences. Many statistical and computational methods have been proposed and applied to detect CNVs based on data that generated by genome analysis platforms. However, most algorithms are computationally intensive with complexity at least $O(n^{2})$, where $n$ is the number of probes in the experiments. Moreover, the theoretical properties of those existing methods are not well understood. A faster and better characterized algorithm is desirable for the ultra high throughput data. In this study, we propose the Screening and Ranking algorithm (SaRa) which can detect CNVs fast and accurately with complexity down to $O(n)$. In addition, we characterize theoretical properties and present numerical analysis for our algorithm.