Chad Tomlinson

A catalog of reference genomes from the human microbiome.

News from the Inner Tube of Life A major initiative by the U.S. National Institutes of Health to sequence 900 genomes of microorganisms that live on the surfaces and orifices of the human body has established standardized protocols and methods for such large-scale reference sequencing. By combining previously accumulated data with new data, Nelson et al. (p. 994) present an initial analysis of 178 bacterial genomes. The sampling so far barely scratches the surface of the microbial diversity found on humans, but the work provides an important baseline for future analyses. Standardized protocols and methods are being established for large-scale sequencing of the microorganisms living on humans. The human microbiome refers to the community of microorganisms, including prokaryotes, viruses, and microbial eukaryotes, that populate the human body. The National Institutes of Health launched an initiative that focuses on describing the diversity of microbial species that are associated with health and disease. The first phase of this initiative includes the sequencing of hundreds of microbial reference genomes, coupled to metagenomic sequencing from multiple body sites. Here we present results from an initial reference genome sequencing of 178 microbial genomes. From 547,968 predicted polypeptides that correspond to the gene complement of these strains, previously unidentified (“novel”) polypeptides that had both unmasked sequence length greater than 100 amino acids and no BLASTP match to any nonreference entry in the nonredundant subset were defined. This analysis resulted in a set of 30,867 polypeptides, of which 29,987 (~97%) were unique. In addition, this set of microbial genomes allows for ~40% of random sequences from the microbiome of the gastrointestinal tract to be associated with organisms based on the match criteria used. Insights into pan-genome analysis suggest that we are still far from saturating microbial species genetic data sets. In addition, the associated metrics and standards used by our group for quality assurance are presented.

Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment

BackgroundAdult house flies, Musca domestica L., are mechanical vectors of more than 100 devastating diseases that have severe consequences for human and animal health. House fly larvae play a vital role as decomposers of animal wastes, and thus live in intimate association with many animal pathogens.ResultsWe have sequenced and analyzed the genome of the house fly using DNA from female flies. The sequenced genome is 691 Mb. Compared with Drosophila melanogaster, the genome contains a rich resource of shared and novel protein coding genes, a significantly higher amount of repetitive elements, and substantial increases in copy number and diversity of both the recognition and effector components of the immune system, consistent with life in a pathogen-rich environment. There are 146 P450 genes, plus 11 pseudogenes, in M. domestica, representing a significant increase relative to D. melanogaster and suggesting the presence of enhanced detoxification in house flies. Relative to D. melanogaster, M. domestica has also evolved an expanded repertoire of chemoreceptors and odorant binding proteins, many associated with gustation.ConclusionsThis represents the first genome sequence of an insect that lives in intimate association with abundant animal pathogens. The house fly genome provides a rich resource for enabling work on innovative methods of insect control, for understanding the mechanisms of insecticide resistance, genetic adaptation to high pathogen loads, and for exploring the basic biology of this important pest. The genome of this species will also serve as a close out-group to Drosophila in comparative genomic studies.

A New Chicken Genome Assembly Provides Insight into Avian Genome Structure

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts.

INTEGRATE: gene fusion discovery using whole genome and transcriptome data

While next-generation sequencing (NGS) has become the primary technology for discovering gene fusions, we are still faced with the challenge of ensuring that causative mutations are not missed while minimizing false positives. Currently, there are many computational tools that predict structural variations (SV) and gene fusions using whole genome (WGS) and transcriptome sequencing (RNA-seq) data separately. However, as both WGS and RNA-seq have their limitations when used independently, we hypothesize that the orthogonal validation from integrating both data could generate a sensitive and specific approach for detecting high-confidence gene fusion predictions. Fortunately, decreasing NGS costs have resulted in a growing quantity of patients with both data available. Therefore, we developed a gene fusion discovery tool, INTEGRATE, that leverages both RNA-seq and WGS data to reconstruct gene fusion junctions and genomic breakpoints by split-read mapping. To evaluate INTEGRATE, we compared it with eight additional gene fusion discovery tools using the well-characterized breast cell line HCC1395 and peripheral blood lymphocytes derived from the same patient (HCC1395BL). The predictions subsequently underwent a targeted validation leading to the discovery of 131 novel fusions in addition to the seven previously reported fusions. Overall, INTEGRATE only missed six out of the 138 validated fusions and had the highest accuracy of the nine tools evaluated. Additionally, we applied INTEGRATE to 62 breast cancer patients from The Cancer Genome Atlas (TCGA) and found multiple recurrent gene fusions including a subset involving estrogen receptor. Taken together, INTEGRATE is a highly sensitive and accurate tool that is freely available for academic use.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

The genome of the vervet (Chlorocebus aethiops sabaeus)

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

The characterization of the Phlebotomus papatasi transcriptome

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re‐emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in‐depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome.

Clonal polymorphism and high heterozygosity in the celibate genome of the Amazon molly

The extreme rarity of asexual vertebrates in nature is generally explained by genomic decay due to absence of meiotic recombination, thus leading to extinction of such lineages. We explore features of a vertebrate asexual genome, the Amazon molly, Poecilia formosa, and find few signs of genetic degeneration but unique genetic variability and ongoing evolution. We uncovered a substantial clonal polymorphism and, as a conserved feature from its interspecific hybrid origin, a 10-fold higher heterozygosity than in the sexual parental species. These characteristics seem to be a principal reason for the unpredicted fitness of this asexual vertebrate. Our data suggest that asexual vertebrate lineages are scarce not because they are at a disadvantage, but because the genomic combinations required to bypass meiosis and to make up a functioning hybrid genome are rarely met in nature.Asexual vertebrates are extremely rare. Here, the authors sequence the genome of the Amazon molly, an asexual fish, and find few signs of genetic degeneration but clonal polymorphism and high heterozygosity, which might explain the success of this species.

The Biomphalaria glabrata DNA methylation machinery displays spatial tissue expression, is differentially active in distinct snail populations and is modulated by interactions with Schistosoma mansoni

Background The debilitating human disease schistosomiasis is caused by infection with schistosome parasites that maintain a complex lifecycle alternating between definitive (human) and intermediate (snail) hosts. While much is known about how the definitive host responds to schistosome infection, there is comparably less information available describing the snail’s response to infection. Methodology/Principle findings Here, using information recently revealed by sequencing of the Biomphalaria glabrata intermediate host genome, we provide evidence that the predicted core snail DNA methylation machinery components are associated with both intra-species reproduction processes and inter-species interactions. Firstly, methyl-CpG binding domain protein (Bgmbd2/3) and DNA methyltransferase 1 (Bgdnmt1) genes are transcriptionally enriched in gonadal compared to somatic tissues with 5-azacytidine (5-AzaC) treatment significantly inhibiting oviposition. Secondly, elevated levels of 5-methyl cytosine (5mC), DNA methyltransferase activity and 5mC binding in pigmented hybrid- compared to inbred (NMRI)- B. glabrata populations indicate a role for the snail’s DNA methylation machinery in maintaining hybrid vigour or heterosis. Thirdly, locus-specific detection of 5mC by bisulfite (BS)-PCR revealed 5mC within an exonic region of a housekeeping protein-coding gene (Bg14-3-3), supporting previous in silico predictions and whole genome BS-Seq analysis of this species’ genome. Finally, we provide preliminary evidence for parasite-mediated host epigenetic reprogramming in the schistosome/snail system, as demonstrated by the increase in Bgdnmt1 and Bgmbd2/3 transcript abundance following Bge (B. glabrata embryonic cell line) exposure to parasite larval transformation products (LTP). Conclusions/Significance The presence of a functional DNA methylation machinery in B. glabrata as well as the modulation of these gene products in response to schistosome products, suggests a vital role for DNA methylation during snail development/oviposition and parasite interactions. Further deciphering the role of this epigenetic process during Biomphalaria/Schistosoma co-evolutionary biology may reveal key factors associated with disease transmission and, moreover, enable the discovery of novel lifecycle intervention strategies.

The Novel Evolution of the Sperm Whale Genome

Abstract The sperm whale, made famous by Moby Dick, is one of the most fascinating of all ocean-dwelling species given their unique life history, novel physiological adaptations to hunting squid at extreme ocean depths, and their position as one of the earliest branching toothed whales (Odontoceti). We assembled the sperm whale (Physeter macrocephalus) genome and resequenced individuals from multiple ocean basins to identify new candidate genes for adaptation to an aquatic environment and infer demographic history. Genes crucial for skin integrity appeared to be particularly important in both the sperm whale and other cetaceans. We also find sperm whales experienced a steep population decline during the early Pleistocene epoch. These genomic data add new comparative insight into the evolution of whales.