Projector 2: contig mapping for efficient gap-closure of prokaryotic genome sequence assemblies

With genome sequencing efforts increasing exponentially, valuable information accumulates on genomic content of the various organisms sequenced. Projector 2 uses (un)finished genomic sequences of an organism as a template to infer linkage information for a genome sequence assembly of a related organism being sequenced. The remaining gaps between contigs for which no linkage information is present can subsequently be closed with direct PCR strategies. Compared with other implementations, Projector 2 has several distinctive features: a user-friendly web interface, automatic removal of repetitive elements (repeat-masking) and automated primer design for gap-closure purposes. Moreover, when using multiple fragments of a template genome, primers for multiplex PCR strategies can also be designed. Primer design takes into account that, in many cases, contig ends contain unreliable DNA sequences and repetitive sequences. Closing the remaining gaps in prokaryotic genome sequence assemblies is thereby made very efficient and virtually effortless. We demonstrate that the use of single or multiple fragments of a template genome (i.e. unfinished genome sequences) in combination with repeat-masking results in mapping success rates close to 100%. The web interface is freely accessible at

BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical “contig” maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning ~10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual “BAC-HAPPY-mapping” to convert BAC landing data into BAC linkage contigs is possible.Giang T. H. Vu, Paul H. Dear, Peter D. S. Caligari and Mike J. Wilkinso

Finishing genomes with limited resources: lessons from an ensemble of microbial genomes

While new sequencing technologies have ushered in an era where microbial genomes can be easily sequenced, the goal of routinely producing high-quality draft and finished genomes in a cost-effective fashion has still remained elusive. Due to shorter read lengths and limitations in library construction protocols, shotgun sequencing and assembly based on these technologies often results in fragmented assemblies. Correspondingly, while draft assemblies can be obtained in days, finishing can take many months and hence the time and effort can only be justified for high-priority genomes and in large sequencing centers. In this work, we revisit this issue in light of our own experience in producing finished and nearly-finished genomes for a range of microbial species in a small-lab setting. These genomes were finished with surprisingly little investments in terms of time, computational effort and lab work, suggesting that the increased access to sequencing might also eventually lead to a greater proportion of finished genomes from small labs and genomics cores

De novo assembly of the olive fruit fly (Bactrocera oleae) genome with linked-reads and long-read technologies minimizes gaps and provides exceptional Y chromosome assembly

Background: The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit agribusiness industry. This is because female flies lay their eggs in the unripe fruits and upon hatching the larvae feed on the fruits thus destroying them. The lack of a high-quality genome and other genomic and transcriptomic data has hindered progress in understanding the fly’s biology and proposing alternative control methods to pesticide use. Results: Genomic DNA was sequenced from male and female Demokritos strain flies, maintained in the laboratory for over 45 years. We used short-, mate-pair-, and long-read sequencing technologies to generate a combined male-female genome assembly (GenBank accession GCA_001188975.2). Genomic DNA sequencing from male insects using 10x Genomics linked-reads technology followed by mate-pair and long-read scaffolding and gap-closing generated a highly contiguous 489 Mb genome with a scaffold N50 of 4.69 Mb and L50 of 30 scaffolds (GenBank accession GCA_001188975.4). RNA-seq data generated from 12 tissues and/or developmental stages allowed for genome annotation. Short reads from both males and females and the chromosome quotient method enabled identification of Y-chromosome scaffolds which were extensively validated by PCR. Conclusions: The high-quality genome generated represents a critical tool in olive fruit fly research. We provide an extensive RNA-seq data set, and genome annotation, critical towards gaining an insight into the biology of the olive fruit fly. In addition, elucidation of Y-chromosome sequences will advance our understanding of the Y-chromosome’s organization, function and evolution and is poised to provide avenues for sterile insect technique approaches

Next Generation Sequencing Technologies for Insect Virus Discovery

Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for de novo assembly of viral sequences, identification of potential viral sequences from BLAST data, and bioinformatics for generating full-length or near full-length viral genome sequences. We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms

Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections

Standard methods for molecular research in Apis mellifera

From studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al. (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees: Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.Fundação para a Ciência e Tecnologi

Whole genome assembly and gap closure of the toxic bloom-forming cyanobacterium Anabaena sp. strain 90

Anabaena is a common member of the phytoplankton in lakes, reservoirs and ponds throughout the world. This is a filamentous, nitrogen-fixing cyanobacterial genus and is frequently present in the lakes of Finland. Anabaena sp. strain 90 was isolated from Lake Vesijärvi and produces microcystins, anabaenopeptilides and anabaenopeptins. A whole genome shotgun sequencing project was undertaken to obtain the complete genome of this organism in order to better understand the physiology and environmental impact of toxic cyanobacteria. This work describes the genome assembly and finishing, the genome structure, and the results of intensive computational analysis of the Anabaena sp. strain 90 genome. Altogether 119,316 sequence reads were generated from 3 genomic libraries with 2, 6 and 40 kb inserts from high throughput Sanger sequencing. The software package Phred/Phrap/Consed was used for whole genome assembly and finishing. A combinatorial PCR method was used to establish relationships between remaining contigs after thorough scaffolding and gap-filling. The final assembly results show that there is a single 4.3 Mb circular chromosome and 4 circular plasmids with sizes of 820, 80, 56 and 20 kb respectively. Together, these 4 plasmids comprise nearly one-fifth of the total genome. Genomic variations in the form of 79 single nucleotide polymorphisms and 3 sequence indels were identified from the assembly results. Sequence analysis revealed that 7.5 percent of the Anabaena sp. strain 90 genome consists of repetitive DNA elements. The genome sequence of Anabaena sp. strain 90 provides a more solid basis for further studies of bioactive compound production, photosynthesis, nitrogen fixation and akinete formation in cyanobacteria

FILAMENTOUS BACTERIOPHAGE ASSOCIATED WITH SHAPING COMMUNITY STRUCTURE AND FITNESS OF INVASIVE VIBRIO PARAHAEMOLYTICUS ST36

Vibrio parahaemolyticus a ubiquitous coastal inhabitant, is the leading cause of bacterial seafood-borne illnesses in the United States. An increasing number of reported cases and rapid expansion into new areas has led to the classification of V. parahaemolyticus as an emergent pathogen. Most strains of V. parahaemolyticus are not virulent; however, the spread of virulent lineages from their native ranges to new locations has contributed drastically to the increase in vibriosis attributed to V. parahaemolyticus in recent years. In the United States (US), sequence type (ST) 36, a virulent strain endemic to the Pacific Northwest (PNW), spread from its native range up and down both coasts of North America even crossed the Atlantic to cause an outbreak in Spain in 2012, Specifically, the North Atlantic coast of the US traditionally did not have a major disease burden due to V. parahaemolyticus; however, the introduction of ST36 and the evolution of local pathogenic lineages have led to a sharp increase in the number of cases traced to product from this region. Here we use genomics and phylogeographic analysis to examine the dynamics of the expansion of ST36 and its subsequent establishment in Northeast coastal waters. The impact of basal acquisition of two unique filamentous bacteriophages by distinct clonal clades within the Northeastern ST36 populations is also explored. We propose that the acquisition of these bacteriophages influenced the fitness of their hosts and enabled the establishment of robust local populations of pathogenic V. parahaemolyticus, contributing greatly to the disease burden in the Northeast. Filamentous bacteriophages are distributed throughout many V. parahaemolyticus populations and may be important drivers of evolution amongst these strains. In direct competition under laboratory conditions, the bacteriophage associated with the Gulf of Maine clonal population, Vipa26, does not impact growth of persistently infected isolates and protects them from superinfection by similar phages. Upon new infection, the growth of susceptible isolates slows dramatically before the integration and down regulation of phage production. qPCR assays for integrated and replicative form of phage elucidate this dynamic during infection. This implicates Vipa26 as a potential sword and shield for this strain, possibly aiding the progenitor of the Gulf of Maine population of ST36 in its subsequent global expansion. Impact of phage on biofilm formation, resistance to predatory grazing and competitive fitness in natural seawater microcosms were also investigated. These studies indicate that phage integration is linked to environmental fitness of ST36 and further investigation into the phage-host relationship is warranted to shed light onto the dynamics of the establishment of novel V. parahaemolyticus populations

Plasmid analysis, comparative genomics and transcriptomics of beer-spoilage lactic acid bacteria emphasizing the role of dissolved carbon dioxide and traditional beer-spoilage markers

Specific isolates of lactic acid bacteria (LAB) are capable of growing in and spoiling beer, and are the cause of product and process contamination, and financial loss for brewers the world over. To date, our understanding of how these contaminants are able to grow in beer is limited to analysis of hop-tolerance mechanisms, with a limited number of putative hop-tolerance genes having been described. In order to demonstrate that these hop-tolerance genes are incomplete descriptors of overall beer-spoilage ability, the transcriptional activity of these genes in two different beer-spoilage related (BSR) LAB isolates, and the prevalence and sequence conservation of hop-tolerance gene horC in BSR LAB with varying beer-spoilage ability is examined. This analysis is followed by work demonstrating that the total plasmid profile of a beer-spoilage LAB, and not just plasmids harboring hop-tolerance genes, contributes to the isolate’s overall beer-spoilage phenotype and highlights redundancy in potential beer-spoilage mechanisms. The next chapter provides evidence that the presence of dissolved CO2 (dCO2) in beer selects for the ability of LAB to spoil packaged beer, and that tolerance to this stress is not correlated with hop-tolerance, indicating that dCO2 stress is an important part of the total beer environment. This is followed by the presentation and analysis of the genome of the rapid beer-spoiling isolate Lactobacillus brevis BSO 464 and subsequent RNA sequencing for this isolate when grown in degassed and gassed beer so as to elucidate which genes are active when grown in beer, and when grown specifically in the presence of dCO2. Global transcriptome sequencing of this L. brevis isolate and Pediococcus claussenii ATCC BAA-344T when each were grown in growth-limiting concentrations of hops was also performed in order to clarify the hop-specific transcriptional response from that of the response when these isolates grow in the total beer environment. Lastly, comparison is made between available genomes of BSR LAB to reveal that the specific brewery environment a BSR LAB is recovered from, influences genetic variability and that comparison within a given LAB species reveals genetic differences that can be exploited as beer-spoilage genetic markers. This comparative analysis reveals that the total plasmid-coding capacity strongly influences individual BSR LAB beer-spoilage phenotype and the environment they are able to grow in. Overall, beer-spoilage ability is shown to be adaptive and acquired incrementally and not solely as a result of the presence of hop-tolerance genes