36 research outputs found
Targeted resequencing of the EGFR and MET genes using the Fluidigm Access Array System and the Roche GS Junior System
No full textShotgun sequencing and assembly of the genome of Geospiza magnirostris – one of the Darwin’s finches
No full text<p>A paper describing this data set is in preparation. Draft text of the paper is below;</p>
<p><strong>Introduction</strong></p>
<p>“Darwin’s Finches” are a model system for the study of various aspects of evolution and development. In 2008 we commenced on a project to sequence the genomes of some of these species – inspired by the (then) upcoming celebration of the 200<sup>th</sup> anniversary of the birth of Charles Darwin (which was in February 2009). The project started with a brief discussion at the AGBT meeting in 2008 and then via an email conversation between Jonathan Eisen and Jason Affourtit about the possibility of a collaboration involving the 454 company (which was looking for projects to highlight the power of it’s then relatively new 454 sequencing machines). After further discussions between Jonathan Eisen, his brother Michael Eisen (who separately had become interested in Darwin’s finches) and people from 454 it was decided that this was a potentially good project for a scientific and marketing collaboration. </p>
<p>In these conversations it was determined that the most likely limiting factor would be access to DNA from the finches. This was largely an issue due to the fact that the Galapagos Islands (where the finches reside) are a National Park in Ecuador and also a World Heritage site. Collection of samples there for any type of research is highly regulated. Thus, Jonathan Eisen made contact with Peter and Rosemary Grant – the most prominent researchers working on the finches – and who Eisen had discussed sequencing the finch genomes in the early 2000s. In that previous conversation it was determined that the sequencing would be too expensive to carry out without a major fundraising effort. However, with the advent of “next generation” sequencing methods such as 454 the total costs of such a project would be much lower. In the conversations with the Grants the Grants offered to ask around to see if anyone had sufficient amounts of DNA (or access to samples), which would be needed for genome library construction. Subsequently they identified Arkhat Abzhanov from Harvard as someone who likely had samples as well as permission to do DNA-based work on them, from many of the finch species.</p>
<p>Abzhanov offered to provide samples from three key species (large ground finch <em>Geospiza magnirostris</em>, large cactus finch <em>G. conirostris</em> and sharp-billed finch <em>G. difficilis</em>) and DNA was sent to Roche-454 for sequencing in July of 2008. In August, the first “test” sequence data was provided from <em>Geospiza magnirostris</em>. A plan was then made to generate additional data and Roche offered to do the sequencing at their center at a steep discount. Funds were raised by Jonathan Eisen, Greg Wray, Monica Riley, and others to pay for the sequencing and over the next year or so, three sequencing bursts were conducted at Roche-454. As the most deeply sequenced species was <em>G. magnirostris</em>, an assembly was generated for this species using Newbler. We report here the results of the sequencing and assembly and present this as a “data paper” in order to make it available to the community. </p>
<p><strong>Methods</strong></p>
<p>DNA isolation: Samples were provided by Arkhat Abzhanov. A genomic DNA samples were taken from individual late stage embryos representing the three species of Darwin’s finches (G. magnirostris, G. conirostris and G. difficilis) collected during a field trip to the island of Genovesa (Galápagos) in 2009. The embryonic trunk tissue was preserved in RNAlater solution (Ambion) and treated as fresh tissue with a commercial genomic DNA preparation kit (QIAGEN Genomic DNA Purification Kit). The quality of the obtained gDNA was checked with a NanoDrop Spectrophotometer (ThermoScientific) and Agilent 2100 Bioanalyzer.</p>
<p>Library construction and sequencing: DNA library construction and sequencing was done at 454-Corporation under the coordination of Timothy Harkins, Jason Affourtit, Clotilde Teiling and Benjamin Boese. DNA libraries were constructed using standard techniques for Roche-454 sequencing. In summary: 3 ug of purified genomic DNA was fractionated into fragments of the targeted size ranges; short adaptors were ligated to each fragments; single stranded fragments created and immobilized onto specifically designed DNA capture beads; the bead-bound library was emulsified with amplification reagents in water in oil mixture resulting in microreactors containing just (ideally) one bead with one unique sample-library fragment; emulsion beads then submitted to PCR amplification; emulsion mixture is then broken while the amplified fragments remain bound to their beads; and the DNA-carrying capture beads are loaded onto a PicoTiterPlate device for sequencing. The device was then loaded into the Genome Sequencer system where individual nucleotides are flowed in a fixed order across the open wells and DNA capture beads; complementary nucleotides to the template strand results in a chemiluminescent signal recorded by the CCD camera of the instrument. Roche-454 software was then used to determine the sequence of ~900,000 reads per instrument run – this is done by analyzing a combination of signal intensity and positional information generated across the PicoTiterPlate device.</p>
<p><strong>Sequencing results</strong></p>
<p>In total twenty-eight long read runs, ninety-six runs on 2.5kbp mate-pair libraries, and forty runs on 5kbp mate-pair libraries were generated. Mate-pair libraries in each size range were constructed multiple times, yielding five mate-pair libraries of approximately 5kbp insert size and an additional five libraries at about 2.5kbp. More detail on the sequencing data is provided in appendix 1.</p>
<p>Assembly of G. magnirostris genome: Data from twenty-eight long read runs, ninety-six runs on 2.5kbp mate-pair libraries, and forty runs on 5kbp mate-pair libraries were combined for the assembly. Mate-pair libraries in each size range were constructed multiple times, yielding five mate-pair libraries of approximately 5kbp insert size and an additional five libraries at about 2.5kbp. Pyrosequencing reads in SFF format were assembled by the Newbler software version 2.3 using the vendor recommended protocol. Briefly, contigs were generated using the long read data, and mate-pair reads were mapped to the contigs and used to link contigs into scaffolds. In total, 24.4 million reads comprising 7.0Gbp were used to form contigs and an additional 4.1 million read pairs were used for scaffolding.</p>
<p>The resulting assembly contains 12958 scaffolds in an estimated genome size of 1254.6Mbp, with a scaffold N50 of 382kbp. The scaffolds comprise 394409 contigs spanning 958.3Mbp. The coverage distribution has a median at 6.5x with a long tail to the right, suggesting that some repeat regions may not be fully resolved. </p>
<p><strong>Conclusion</strong></p>
<p>We are currently carrying out a more detailed analysis of the genome assembly but wanted to release the data to the community and thus have written this “data” paper.</p>
<p><strong>Acknowledgements</strong></p>
<p>We acknowledge the following people: Peter and Rosemary Grant for helping initiate the project, Greg Wray and Peg Riley for financial support, Russell Neches for assistance with some sequencing analyses, and Brian Desany, Karin Frederickson, Courtney Brady and Take Ogawa for help with communications with Roche.</p>
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Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture
No full textDomestication and divergence of Saccharomyces cerevisiae beer yeasts
No full textWhereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains
Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity
No full textEukaryotic microbes (protists) residing in the vertebrate gut influence host health and disease, but their diversity and distribution in healthy hosts is poorly understood. Protists found in the gut are typically considered parasites, but many are commensal and some are beneficial. Further, the hygiene hypothesis predicts that association with our co-evolved microbial symbionts may be important to overall health. It is therefore imperative that we understand the normal diversity of our eukaryotic gut microbiota to test for such effects and avoid eliminating commensal organisms. We assembled a dataset of healthy individuals from two populations, one with traditional, agrarian lifestyles and a second with modern, westernized lifestyles, and characterized the human eukaryotic microbiota via high-throughput sequencing. To place the human gut microbiota within a broader context our dataset also includes gut samples from diverse mammals and samples from other aquatic and terrestrial environments. We curated the SILVA ribosomal database to reflect current knowledge of eukaryotic taxonomy and employ it as a phylogenetic framework to compare eukaryotic diversity across environment. We show that adults from the non-western population harbor a diverse community of protists, and diversity in the human gut is comparable to that in other mammals. However, the eukaryotic microbiota of the western population appears depauperate. The distribution of symbionts found in mammals reflects both host phylogeny and diet. Eukaryotic microbiota in the gut are less diverse and more patchily distributed than bacteria. More broadly, we show that eukaryotic communities in the gut are less diverse than in aquatic and terrestrial habitats, and few taxa are shared across habitat types, and diversity patterns of eukaryotes are correlated with those observed for bacteria. These results outline the distribution and diversity of microbial eukaryotic communities in the mammalian gut and across environments
RAST_Annotations
No full textRAST-generated annotations for all genomes included in the analyses in this manuscript
Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture
No full textMany microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite's genome
