Assembly Annotation Research Articles

An ecological model organism flies into the genomics era.

Despite the very rapid 'genomicization' of the field of Molecular Ecology in recent years, there have been relatively few annotated whole-genome assemblies of nonmodel organisms published. Instead, molecular ecologists have more frequently utilized next-generation sequencing technologies to develop genome-wide markers or to generate transcriptome data. Whole-genome assemblies are more expensive and require considerable computational resources and bioinformatic expertise. However, the availability of an annotated genome offers exciting opportunities to address fundamental questions in ecology and evolution that are difficult to address with moderate sets of markers or by transcriptome sequencing. Such questions include elucidating the roles of natural and sexual selection in shaping diversity, determining the roles of regulatory and protein-coding change in the evolution of traits, and determining the genomic architecture of sex-specific trait variation. Arguably, these questions are most tractable--and most interesting--in well-characterized species for which there is already some knowledge of natural and sexual selection, and of the traits that are most likely to link to fitness. In this issue, Mueller et al. (2016) present the assembly and annotation of the genome of the blue tit (Cyanistes caeruleus), a model ecological species. In addition, by sequencing the transcriptome of male and female blue tits, the authors identify and annotate sex-biased gene expression and conclude that noncoding RNA genes are likely to play a significant role in sex-biased expression. By making their assembly and annotation publically available and accessible via a genome browser, Mueller et al. (2016) offer exciting possibilities for further research into the genomic basis of adaptation, and investigation of the roles of natural and sexual selection, in this well-studied ecological model species.

Deep Sequencing of the Fruit Transcriptome and Lipid Accumulation in a Non-Seed Tissue of Chinese Tallow, a Potential Biofuel Crop.

Chinese tallow (Triadica sebifera) is a valuable oilseed-producing tree that can grow in a variety of conditions without competing for food production, and is a promising biofuel feedstock candidate. The fruits are unique in that they contain both saturated and unsaturated fat present in the tallow and seed layer, respectively. The tallow layer is poorly studied and is considered only as an external fatty deposition secreted from the seed. In this study we show that tallow is in fact a non-seed cellular tissue capable of triglyceride synthesis. Knowledge of lipid synthesis and storage mechanisms in tissues other than seed is limited but essential to generate oil-rich biomass crops. Here, we describe the annotated transcriptome assembly generated from the fruit coat, tallow and seed tissues of Chinese tallow. The final assembly was functionally annotated, allowing for the identification of candidate genes and reconstruction of lipid pathways. A tallow tissue-specific paralog for the transcription factor gene WRINKLED1 (WRI1) and lipid droplet-associated protein genes, distinct from those expressed in seed tissue, were found to be active in tallow, underpinning the mode of oil synthesis and packaging in this tissue. Our data have established an excellent knowledge base that can provide genetic and biochemical insights for engineering non-seed tissues to accumulate large amounts of oil. In addition to the large data set of annotated transcripts, the study also provides gene-based simple sequence repeat and single nucleotide polymorphism markers.

A Publishing Method of Lightweight Three-Dimensional Assembly Instruction for Complex Products

In order to accurately guide on-site workers to quickly accomplish the assembly job of complex products, and reduce the deployment cost of assembly instruction, we propose a publishing method of lightweight 3D assembly instruction for complex products. In this paper, the key frames of assembly motion and the 3D technical annotations in the lightweight model are mapped to the time-based assembly process. Then, the annotated lightweight model and assembly process information are integrated and published into a single 3D assembly instruction document. An assembly instruction publishing example of satellite antenna feed component shows that the lightweight 3D assembly instruction is well instructive and affordable because it provides the interactive simulation of assembly process and time-based display of assembly technical annotations without using expensive computer-aided design (CAD) systems, graphics workstations, or virtual reality equipments. This method gives a full play to the advantages of model-based definition technology and lightweight model, and fills the gap between the process planning and the instruction publishing in the 3D virtual manufacturing environment.

The coelomic epithelium transcriptome from a clonal sea star, Coscinasterias muricata

Coscinasterias is a cosmopolitan genus of large asteroid sea stars with the ability of somatic fission as a clonal reproductive strategy. During fission, the animals tear themselves apart across their central disc, where the lost body parts are regenerated afterwards. Here, we have sequenced and subsequently analysed the transcriptome of the coelomic epithelium of a clonal Coscinasterias muricata specimen from New Zealand. Out of the total 389,768 raw reads, 11,344 contigs were assembled and grouped into functions. Raw read and assembled contig sequences are available at NCBI (BioSample: SAMN03371637), while the annotated assembly can be accessed through the project transcriptome browser (compgen.bio.ub.edu/gbrowse/starfish_transcriptome/). Our data is valuable for future detailed exploration of the coelomic epithelium functions as well as for a better understanding of sea star physiology.

Annotation of suprachromosomal families reveals uncommon types of alpha satellite organization in pericentromeric regions of hg38 human genome assembly.

Centromeric alpha satellite (AS) is composed of highly identical higher-order DNA repetitive sequences, which make the standard assembly process impossible. Because of this the AS repeats were severely underrepresented in previous versions of the human genome assembly showing large centromeric gaps. The latest hg38 assembly (GCA_000001405.15) employed a novel method of approximate representation of these sequences using AS reference models to fill the gaps. Therefore, a lot more of assembled AS became available for genomic analysis. We used the PERCON program previously described by us to annotate various suprachromosomal families (SFs) of AS in the hg38 assembly and presented the results of our primary analysis as an easy-to-read track for the UCSC Genome Browser. The monomeric classes, characteristic of the five known SFs, were color-coded, which allowed quick visual assessment of AS composition in whole multi-megabase centromeres down to each individual AS monomer. Such comprehensive annotation of AS in the human genome assembly was performed for the first time. It showed the expected prevalence of the known major types of AS organization characteristic of the five established SFs. Also, some less common types of AS arrays were identified, such as pure R2 domains in SF5, apparent J/R and D/R mixes in SF1 and SF2, and several different SF4 higher-order repeats among reference models and in regular contigs. No new SFs or large unclassed AS domains were discovered. The dataset reveals the architecture of human centromeres and allows classification of AS sequence reads by alignment to the annotated hg38 assembly. The data were deposited here: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&hgt.customText=https://dl.dropboxusercontent.com/u/22994534/AS-tracks/human-GRC-hg38-M1SFs.bed.bz2.

Complete Genome Assembly of Staphylococcus epidermidis AmMS 205.

Staphylococcus epidermidis causes a large number of catheter-related sepsis infections annually in the United States. We present the 2.54-Mbp complete genome assembly of reference strain S. epidermidis AmMS 205, including a single 37.7-kbp plasmid. The annotated assembly is available in GenBank under accession numbers CP009046 and CP009047.

Genome Sequence Analysis of the Biogenic Amine-Producing Strain Lactococcus lactis subsp. cremoris CECT 8666 (Formerly GE2-14).

We here report a 2,801,031-bp annotated draft assembly for the Lactococcus lactis subsp. cremoris GE2-14 genome. This dairy strain produces the biogenic amine putrescine. This sequence may help identify the mechanisms regulating putrescine biosynthesis and throw light on ways to reduce its presence in fermented foods.

Whole-Genome Sequence of Listeria monocytogenes Type Strain 53 XXIII.

Listeria monocytogenes causes the food-borne illness listeriosis that primarily infects “vulnerable” groups (e.g., elderly adults, pregnant women, very young children, and the immunocompromised). We sequenced the genome of L. monocytogenes XXIII (ATCC 15313) and assembled it into a single scaffold (three contigs) and deposited the annotated assembly into GenBank as accession no. JOOX00000000.

Genome Sequence Analysis of the Biogenic Amine-Degrading Strain Lactobacillus casei 5b

We here report a 3.02-Mbp annotated draft assembly of the Lactobacillus casei 5b genome. The sequence of this biogenic amine-degrading dairy isolate may help identify the mechanisms involved in the catabolism of biogenic amines and perhaps shed light on ways to reduce the presence of these toxic compounds in food.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BackgroundCork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.ResultsWe generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.ConclusionsThis genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Functional Immunomics of the Squash Bug, Anasa tristis (De Geer) (Heteroptera: Coreidae).

The Squash bug, Anasa tristis (De Geer), is a major piercing/sucking pest of cucurbits, causing extensive damage to plants and fruits, and transmitting phytopathogens. No genomic resources to facilitate field and laboratory studies of this pest were available; therefore the first de novo exome for this destructive pest was assembled. RNA was extracted from insects challenged with bacterial and fungal immunoelicitors, insects fed on different cucurbit species, and insects from all life stages from egg to adult. All treatments and replicates were separately barcoded for subsequent analyses, then pooled for sequencing in a single lane using the Illumina HiSeq2000 platform. Over 211 million 100-base tags generated in this manner were trimmed, filtered, and cleaned, then assembled into a de novo reference transcriptome using the Broad Institute Trinity assembly algorithm. The assembly was annotated using NCBIx NR, BLAST2GO, KEGG and other databases. Of the >130,000 total assemblies 37,327 were annotated identifying the sequences of candidate gene silencing targets from immune, endocrine, reproductive, cuticle, and other physiological systems. Expression profiling of the adult immune response was accomplished by aligning the 100-base tags from each biological replicate from each treatment and controls to the annotated reference assembly of the A. tristis transcriptome.

Agalma: an automated phylogenomics workflow

BackgroundIn the past decade, transcriptome data have become an important component of many phylogenetic studies. They are a cost-effective source of protein-coding gene sequences, and have helped projects grow from a few genes to hundreds or thousands of genes. Phylogenetic studies now regularly include genes from newly sequenced transcriptomes, as well as publicly available transcriptomes and genomes. Implementing such a phylogenomic study, however, is computationally intensive, requires the coordinated use of many complex software tools, and includes multiple steps for which no published tools exist. Phylogenomic studies have therefore been manual or semiautomated. In addition to taking considerable user time, this makes phylogenomic analyses difficult to reproduce, compare, and extend. In addition, methodological improvements made in the context of one study often cannot be easily applied and evaluated in the context of other studies.ResultsWe present Agalma, an automated tool that constructs matrices for phylogenomic analyses. The user provides raw Illumina transcriptome data, and Agalma produces annotated assemblies, aligned gene sequence matrices, a preliminary phylogeny, and detailed diagnostics that allow the investigator to make extensive assessments of intermediate analysis steps and the final results. Sequences from other sources, such as externally assembled genomes and transcriptomes, can also be incorporated in the analyses. Agalma is built on the BioLite bioinformatics framework, which tracks provenance, profiles processor and memory use, records diagnostics, manages metadata, installs dependencies, logs version numbers and calls to external programs, and enables rich HTML reports for all stages of the analysis. Agalma includes a small test data set and a built-in test analysis of these data. In addition to describing Agalma, we here present a sample analysis of a larger seven-taxon data set. Agalma is available for download at https://bitbucket.org/caseywdunn/agalma.ConclusionsAgalma allows complex phylogenomic analyses to be implemented and described unambiguously as a series of high-level commands. This will enable phylogenomic studies to be readily reproduced, modified, and extended. Agalma also facilitates methods development by providing a complete modular workflow, bundled with test data, that will allow further optimization of each step in the context of a full phylogenomic analysis.

De novo assembly of the pennycress (Thlaspi arvense) transcriptome provides tools for the development of a winter cover crop and biodiesel feedstock

Field pennycress (Thlaspi arvense L.) has potential as an oilseed crop that may be grown during fall (autumn) and winter months in the Midwestern United States and harvested in the early spring as a biodiesel feedstock. There has been little agronomic improvement in pennycress through traditional breeding. Recent advances in genomic technologies allow for the development of genomic tools to enable rapid improvements to be made through genomic assisted breeding. Here we report an annotated transcriptome assembly for pennycress. RNA was isolated from representative plant tissues, and 203 million unique Illumina RNA-seq reads were produced and used in the transcriptome assembly. The draft transcriptome assembly consists of 33 873 contigs with a mean length of 1242 bp. A global comparison of homology between the pennycress and Arabidopsis transcriptomes, along with four other Brassicaceae species, revealed a high level of global sequence conservation within the family. The final assembly was functionally annotated, allowing for the identification of putative genes controlling important agronomic traits such as flowering and glucosinolate metabolism. Identification of these genes leads to testable hypotheses concerning their conserved function and to rational strategies to improve agronomic properties in pennycress. Future work to characterize isoform variation between diverse pennycress lines and develop a draft genome sequence for pennycress will further direct trait improvement.

Transcriptome analysis and gene expression atlas for Panicum hallii var. filipes, a diploid model for biofuel research

Panicum hallii is an emerging model for genetic studies of agronomic traits in Panicum, presenting a tractable diploid alternative study system to the tetra- or octaploid biofuel crop switchgrass (Panicum virgatum). To characterize the gene complement in P.hallii var. filipes and enable gene expression analysis in this system we sequenced, assembled, and annotated the transcriptome. Over 300 Mb of normalized cDNA prepared from multiple tissues and treatments was sequenced using 454-Titanium, producing an annotated assembly including 15 422 unique gene names. Comparison with other grass genomes identified putative P.hallii homologs for >14 000 previously characterized genes. We also developed an atlas of gene expression across tissues and stages using RNA-Seq (the quantitative analysis of short cDNA reads). SOLiD sequencing and quantitative analysis of more than 40 million cDNA tags identified substantial variation in expression profiles among tissues, consistent with known functional differences. Putative homologs were found for all enzymes in the phenylpropanoid pathway leading to lignin biosynthesis, including genes with known effects on biomass conversion efficiency. The resources developed here will enable studies of the genes underlying variation in cell wall composition, drought tolerance, and biomass production in Panicum.

The Comprehensive Phytopathogen Genomics Resource: a web-based resource for data-mining plant pathogen genomes.

The Comprehensive Phytopathogen Genomics Resource (CPGR) provides a web-based portal for plant pathologists and diagnosticians to view the genome and trancriptome sequence status of 806 bacterial, fungal, oomycete, nematode, viral and viroid plant pathogens. Tools are available to search and analyze annotated genome sequences of 74 bacterial, fungal and oomycete pathogens. Oomycete and fungal genomes are obtained directly from GenBank, whereas bacterial genome sequences are downloaded from the A Systematic Annotation Package (ASAP) database that provides curation of genomes using comparative approaches. Curated lists of bacterial genes relevant to pathogenicity and avirulence are also provided. The Plant Pathogen Transcript Assemblies Database provides annotated assemblies of the transcribed regions of 82 eukaryotic genomes from publicly available single pass Expressed Sequence Tags. Data-mining tools are provided along with tools to create candidate diagnostic markers, an emerging use for genomic sequence data in plant pathology. The Plant Pathogen Ribosomal DNA (rDNA) database is a resource for pathogens that lack genome or transcriptome data sets and contains 131 755 rDNA sequences from GenBank for 17 613 species identified as plant pathogens and related genera.Database URL: http://cpgr.plantbiology.msu.edu.

The quest for an Indian blood law as of blood transfusion services regulatory framework

Background:Blood transfusion services are a vital part of the national health delivery system. The responsibility for ensuring a continuous supply of blood rests with health administrators, who need to galvanize entire communities towards regular and non-remunerated blood donation.Objective:The present study aimed to examine the prevailing global regulations and practices related to blood transfusion and press the case for a dedicated blood law in India.Materials and Methods:We attempted a comprehensive, annotated assembly of published studies on blood transfusion services in India.Data ion and Synthesis:Laws related to blood transfusion services exist in India as a part of the Drugs and Cosmetics Law. In the developed world, most blood donors are unpaid volunteers who give blood for a community supply. In order to augment safe blood transfusion services in India, we have to develop operational legal guidelines on recruitment and retention of voluntary blood donors to direct related organizations for this imperative activity.Conclusion:Several factors, such as political will and a professional and ethical approach can help in formulating a common vision, building trust, by providing optimum information towards a social movement for the rational blood transfusion services. We have to come together for a dedicated blood law in order to improve the quality of blood transfusion services in India.

A preliminary comparison of the mouse and human genomes

Accurate annotated assemblies of the mouse and human genomes enable a detailed comparison of the organization and evolution of the two genomes. We have completed several assemblies of both the mouse, with and without public data, and human genomes. Analysis of these assemblies suggests the mouse genome is about 10% smaller than the human genome primarily because of a difference in the content of repetitive DNA between the two genomes. More than 300,000 positions in these two genomes can be aligned with one another based on short segments of sequence similarity. These conserved segments significantly enhance the resolution of the resultant comparative maps and can be used to divide the genomes into regions of conserved-shared synteny. The genes found in such regions are highly conserved as is their relative order and orientation. Comparison of the human and mouse genome is expected to be key to deciphering the important biological information encoded in the mammalian genome. A prerequisite to comparing complex genomes such as those of mouse and human is the availability of annotated assemblies of both genomes that are comparable in quality and completeness. Since February 2001, we have assembled, annotated and delivered to our subscribers two versions of the human genome and two versions of the mouse genome. A third assembly of the human genome is being completed and will be delivered by fall of 2002. These annotated assemblies provide the starting materials for the genome-wide comparisons of the mouse and human reported here. We will begin with a description of the first Celera whole genome assembly of the mouse to provide a general basis of the quality and completeness of these data and then will report the results of a preliminary comparison between these two genomes.

The prostate expression database (PEDB): status and enhancements in 2000.

The Prostate Expression Database (PEDB) is an online resource designed to access and analyze gene expression information derived from the human prostate. PEDB archives >55 000 expressed sequence tags (ESTs) from 43 cDNA libraries in a curated relational database that provides detailed library information including tissue source, library construction methods, sequence diversity and sequence abundance. The differential expression of each EST species can be viewed across all libraries using a Virtual Expression Analysis Tool (VEAT), a graphical user interface written in Java for intra- and inter-library species comparisons. Recent enhancements to PEDB include: (i) the functional categorization of annotated EST assemblies using a classification scheme developed at The Institute for Genome Research; (ii) catalogs of expressed genes in specific prostate tissue sources designated as transcriptomes; and (iii) the addition of prostate proteome information derived from two-dimensional electrophoreses and mass spectrometry of prostate cancer cell lines. PEDB may be accessed via the WWW at http://www.mbt.washington.edu/PEDB/