Brett Chapman

A chromosome conformation capture ordered sequence of the barley genome

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes.

BackgroundEuphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds.ResultsIn this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (> = 75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin.ConclusionThe resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients.

The complete genome sequence of the pathogenic intestinal spirochete Brachyspira pilosicoli and comparison with other Brachyspira genomes

Background The anaerobic spirochete Brachyspira pilosicoli colonizes the large intestine of various species of birds and mammals, including humans. It causes “intestinal spirochetosis”, a condition characterized by mild colitis, diarrhea and reduced growth. This study aimed to sequence and analyse the bacterial genome to investigate the genetic basis of its specialized ecology and virulence. Methodology/Principal Findings The genome of B. pilosicoli 95/1000 was sequenced, assembled and compared with that of the pathogenic Brachyspira hyodysenteriae and a near-complete sequence of Brachyspira murdochii. The B. pilosicoli genome was circular, composed of 2,586,443 bp with a 27.9 mol% G+C content, and encoded 2,338 genes. The three Brachyspira species shared 1,087 genes and showed evidence of extensive genome rearrangements. Despite minor differences in predicted protein functional groups, the species had many similar features including core metabolic pathways. Genes distinguishing B. pilosicoli from B. hyodysenteriae included those for a previously undescribed bacteriophage that may be useful for genetic manipulation, for a glycine reductase complex allowing use of glycine whilst protecting from oxidative stress, and for aconitase and related enzymes in the incomplete TCA cycle, allowing glutamate synthesis and function of the cycle during oxidative stress. B. pilosicoli had substantially fewer methyl-accepting chemotaxis genes than B. hyodysenteriae and hence these species are likely to have different chemotactic responses that may help to explain their different host range and colonization sites. B. pilosicoli lacked the gene for a new putative hemolysin identified in B. hyodysenteriae WA1. Both B. pilosicoli and B. murdochii lacked the rfbBADC gene cluster found on the B. hyodysenteriae plasmid, and hence were predicted to have different lipooligosaccharide structures. Overall, B. pilosicoli 95/1000 had a variety of genes potentially contributing to virulence. Conclusions/Significance The availability of the complete genome sequence of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence.

The genetics of symbiotic nitrogen fixation: Comparative genomics of 14 rhizobia strains by resolution of protein clusters

The symbiotic relationship between legumes and nitrogen fixing bacteria is critical for agriculture, as it may have profound impacts on lowering costs for farmers, on land sustainability, on soil quality, and on mitigation of greenhouse gas emissions. However, despite the importance of the symbioses to the global nitrogen cycling balance, very few rhizobial genomes have been sequenced so far, although there are some ongoing efforts in sequencing elite strains. In this study, the genomes of fourteen selected strains of the order Rhizobiales, all previously fully sequenced and annotated, were compared to assess differences between the strains and to investigate the feasibility of defining a core ‘symbiome’—the essential genes required by all rhizobia for nodulation and nitrogen fixation. Comparison of these whole genomes has revealed valuable information, such as several events of lateral gene transfer, particularly in the symbiotic plasmids and genomic islands that have contributed to a better understanding of the evolution of contrasting symbioses. Unique genes were also identified, as well as omissions of symbiotic genes that were expected to be found. Protein comparisons have also allowed the identification of a variety of similarities and differences in several groups of genes, including those involved in nodulation, nitrogen fixation, production of exopolysaccharides, Type I to Type VI secretion systems, among others, and identifying some key genes that could be related to host specificity and/or a better saprophytic ability. However, while several significant differences in the type and number of proteins were observed, the evidence presented suggests no simple core symbiome exists. A more abstract systems biology concept of nitrogen fixing symbiosis may be required. The results have also highlighted that comparative genomics represents a valuable tool for capturing specificities and generalities of each genome.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. ‘Morex’ was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).

High-throughput parallel proteogenomics: A bacterial case study

In recent years, a new paradigm for genome annotation has emerged, termed proteogenomics, that leverages peptide MS to annotate a genome. This is achieved by mapping peptides to a six‐frame translation of a genome, including available splice databases, which may suggest refinements to gene models. Using this approach, it is possible to refine gene regions such as exon boundaries, novel genes, gene boundaries, frame shifts, reverse strands, translated UTRs, and novel splice junctions. One of the challenges of proteogenomics is how best to (1) tackle assigning confidence to any resulting annotation and (2) apply these gene model refinements, either through manual annotation or through an automated process via training gene prediction tools. This is not a straightforward process, as many gene prediction tools have their defined suitability for niche genomes (either eukaryotic or prokaryotic) trained on and refined with model organisms such as Arabidopsis thaliana and Escherichia coli, and varying degrees of features that can leverage the use of external evidence. In this study, we outline a suitable approach toward preprocessing mass spectra and optimizing the MS/MS search for a given dataset. We also discuss future challenges, which continue to pose a problem in the field of proteogenomics, and better strategies to successfully tackle them with, using existing tools. We use Bradyrhizobium diazoefficiens (Nitrogen‐fixing bacteria), with a 9.1 Mb genome as a case study, utilizing the latest in second‐generation proteogenomics tools with multiple gene models for cross‐validation of proteogenomics annotations.

Plant Proteogenomics: From Protein Extraction to Improved Gene Predictions

Historically many genome annotation strategies have lacked experimental evidence at the protein level, which and have instead relied heavily on ab initio gene prediction tools, which consequently resulted in many incorrectly annotated genomic sequences. Proteogenomics aims to address these issues using mass spectrometry (MS)-based proteomics, genomic mapping, and providing statistical significance measures such as false discovery rates (FDRs) to validate the mapped peptides. Presented here is a tool capable of meeting this goal, the UCSD proteogenomic pipeline, which maps peptide-spectrum matches (PSMs) to the genome using the Inspect MS/MS database search tool and assigns a statistical significance to the match using a target-decoy search approach to assign estimated FDRs. This pipeline also provides the option of using a more reliable approach to proteogenomics by determining the precise false-positive rates (FPRs) and p-values of each PSM by calculating their spectral probabilities and rescoring each PSM accordingly. In addition to the protein prediction challenges in the rapidly growing number of sequenced plant genomes, it is difficult to extract high-quality protein samples from many plant species. For that reason, this chapter contains methods for protein extraction and trypsin digestion that reliably produce samples suitable for proteogenomic analysis.

Metabolomics – The combination of analytical biochemistry, biology, and informatics

The combination of analytical biochemistry to measure the metabolic complement with sophisticated informatics, bioinformatics, and statistics makes up the newest of the ‘omics fields called metabolomics. Metabolites are characterized by a diverse chemistry and therefore require the application of numerous analytical approaches for their extraction, separation, detection, and quantification. In the past decade, the technologies have improved substantially allowing the analysis of thousands of compounds simultaneously. However, this has led to the current bottleneck in metabolomics that is how to extract information from raw data of many different analytical platforms and the subsequent appropriate analysis in a biological context. Before any statistical analysis can be performed on the resulting high-density data sets, a number of preprocessing steps including peak finding, integration, filtering, normalization, and transformation are required. In this article, we aim to summarize the state of the art of metabolomics technologies from both an analytical and a bioinformatics point of view. We present the challenges currently faced by metabolomics researchers and provide the readers with potential approaches to address those challenges.

Gene-enriched draft genome of the cattle tick Rhipicephalus microplus: assembly by the hybrid Pacific Biosciences/Illumina approach enabled analysis of the highly repetitive genome

The genome of the cattle tick Rhipicephalus microplus, an ectoparasite with global distribution, is estimated to be 7.1Gbp in length and consists of approximately 70% repetitive DNA. We report the draft assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genome. Our hybrid approach produced an assembly consisting of 2.0Gbp represented in 195,170 scaffolds with a N50 of 60,284bp. The Rmi v2.0 assembly is 51.46% repetitive with a large fraction of unclassified repeats, short interspersed elements, long interspersed elements and long terminal repeats. We identified 38,827 putative R. microplus gene loci, of which 24,758 were protein coding genes (≥100 amino acids). OrthoMCL comparative analysis against 11 selected species including insects and vertebrates identified 10,835 and 3,423 protein coding gene loci that are unique to R. microplus or common to both R. microplus and Ixodes scapularis ticks, respectively. We identified 191 microRNA loci, of which 168 have similarity to known miRNAs and 23 represent novel miRNA families. We identified the genomic loci of several highly divergent R. microplus esterases with sequence similarity to acetylcholinesterase. Additionally we report the finding of a novel cytochrome P450 CYP41 homolog that shows similar protein folding structures to known CYP41 proteins known to be involved in acaricide resistance.

Plant proteogenomics: Improvements to the grapevine genome annotation

Grapevine is an important perennial fruit to the wine industry, and has implications for the health industry with some causative agents proven to reduce heart disease. Since the sequencing and assembly of grapevine cultivar Pinot Noir, several studies have contributed to its genome annotation. This new study further contributes toward genome annotation efforts by conducting a proteogenomics analysis using the latest genome annotation from CRIBI, legacy proteomics dataset from cultivar Cabernet Sauvignon and a large RNA‐seq dataset. A total of 341 novel annotation events are identified consisting of five frame‐shifts, 37 translated UTRs, 15 exon boundaries, one novel splice, nine novel exons, 159 gene boundaries, 112 reverse strands, and one novel gene event in 213 genes and 323 proteins. From this proteogenomics evidence, the Augustus gene prediction tool predicted 52 novel and revised genes (54 protein isoforms), 11 genes of which are associated with key traits such as stress tolerance and floral and fruity wine characteristics. This study also highlights a likely over‐assembly with the genome, particularly on chromosome 7.