Posts Tagged ‘454 sequencing’

Thelephoroid Fungi

September 5th, 2010

Thelephora terrestrisWe have just received 500,000 454 TTN ESTs of a series of ectomycorrhizal thelephoroid fungi: Thelephora terrestris, Tomentellopsis submollis and Tomentella stuposa. from the Genoscope. T. terrestris (Earthfan) is a very common ‘contaminant’ of seedlings grown in greenhouses and forest nurseries. The Thelephoraceae are one of the most abundant ectomycorrhizal basidiomycete group in boreal and temperate forests, but they often form fruiting bodies on dead woods. It remains to be determined whether their genome and transcriptome reflect this dual lifestyle.

Photo: Earthfan fruiting bodies © F Martin

ECOFINDERS

May 30th, 2010

CB055265Great news!!! To increase the understanding of the role of soil biodiversity in ecosystem functioning, the European Commission (EC) awarded €7 million to our research project ECOFINDERS. This four year project, coordinated by INRA, aims to support European Union soil policy making by providing the necessary tools to design and implement strategies for sustainable use of soils.

The project will include:

  • Characterisation of the biodiversity of European soils and the normal operating range (NOR) according to soil types, threats, climatic zone and land use,
  • Determination of relationships between soil biodiversity, functioning and ecosystem services,
  • Quantification of the economic values of soil ecosystem services,
  • Evaluation of the impacts of human activities on soil biodiversity, functioning and services,
  • Design of policy-relevant and cost-effective indicators for monitoring soil biodiversity, functioning and ecosystem services.

To reach this overall aim, the project will pursue the following:

  • Describe the diversity of soil organisms (microorganisms and fauna) by using nextgen sequencing,
  • Decipher their interactions through trophic food webs,
  • Determine the role played by soil organisms in soil functioning and major ecosystem services: nutrient retention, carbon storage, water retention, soil structure regulation, resistance to pests and diseases, and regulation of above-ground diversity,
  • Assess the stability and resilience of ecosystems against threats in relation to their biodiversity: soil erosion and physical degradation, decline in organic content, loss of soil biodiversity, and soil contamination.

The 22 consortium partners will:

  • Develop and standardise phenotypic tools and procedures to measure the faunal biodiversity,
  • Design molecular methods to characterise the faunal diversity calibrated upon phenotypic traits,
  • Customise functional tools and methods to determine the functional diversity of fauna,
  • Establish high-throughput molecular assays for assessing microbial and faunal biodiversity,
  • Design, develop and establish a database aimed at mapping the European soil biodiversity and threats,
  • Establish cost-effective bioindicators to measure microbial and faunal diversity, their associated functions and the resulting ecosystem services,
  • Evaluate the economic added-value brought by these bioindicators in assessing the consequences of soil management policy for soil biodiversity and functioning,
  • Implement effective dissemination strategies to transfer the project knowledge and tools to soil stakeholders, notably but not exclusively regional, national and European policy-makers, and inform the general public about the issues associated with the sustainability of soil biodiversity.

My lab will focus on developing 454-based genotyping to survey the microbial communities — hundreds of creeping subterranean bugs will ended up in digits. Our on-going analysis of forest soil metagenomes will likely feed this large scale multi-year project.

Next-Generation Sequencing of Sordaria Genome

May 1st, 2010

sordariaAs I reported earlier, the genome of the filamentous pathogenic fungus Grosmannia clavigera has been assembled from a combination of Sanger, 454, and Solexa sequence data.  The genome of the giant panda (Ailuropoda melanoleura) — specifically of the female Beijing Olympics mascot Jingjing — has also been determined using short-read Illumina sequencing technology, “un tour de force” for such a complex genome. This was the first reported de novo assembly of a large mammalian genome achieved using next-generation sequencing methods. These two studies demonstrated the feasibility for using next-generation sequencing technologies (454 & Illumina) for accurate, cost-effective and rapid de novo assembly of eukaryotic genomes. In a recent issue of PLoS Genetics, Nowrousian et al. published the de novo assembly of the 40 Mb genome of Sordaria macrospora from short sequence reads. They generated 3.4 Gb of sequence data from four lanes from a 300 bp library and three lanes from a 500 bp library using the Illumina GA. In addition, 415 Mb of sequence data were produced by 454 sequencing. Assembly of the Solexa reads only as well as the combined Solexa and 454 reads was carried out with the Velvet assembler.

Diego Martinez and Mary Anne Nelson discuss the technical merits of this work in their Perspective paper in PLoS Genetics.

The natural habitat of the saprobic S. macrospora is herbivore dung. With 10,789 predicted genes, the gene repertoire in S. macrospora is similar to that of N. crassaS. macrospora harbors duplications of several genes involved in self/nonself-recognition and contains more polyketide biosynthesis genes than N. crassa. One putative polyketide biosynthesis (PKS) cluster might have been acquired through horizontal gene transfer (HGT) from a distantly related ascomycete group. This finding supports recent suggestions that HGT is widespread in fungi both for the transfer of single genes, clustered genes like PKS genes, or even larger stretches of DNA up to whole chromosomes as was found in the phytopathogenic fungus Nectria haematococca. The S. macrospora genome contains even fewer transposable elements than its closest relative, Neurospora crassa, despite the absence of active RIP.

Nowrousian M, Stajich JE, Chu M, Engh I, Espagne E, et al. (2010) De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis. PLoS Genet 6(4): e1000891. doi:10.1371/journal.pgen.1000891.

Li et al. (2010) The sequence and de novo assembly of the giant panda genome. Nature 463, 311-317.

Martinez DA, Nelson MA (2010) The Next Generation Becomes the Now Generation. PLoS Genet 6(4): e1000906.

Photo: S. macrocarpa protoperithecium (Nowrousian et al. 2010. PLoS Genetics).).

One Bacterial Cell, One Complete Genome

May 1st, 2010

90% of microbial bugs are eluding current culturing attempts. Sequencing of single cells is a novel culture-independent approach, which enables access to the genetic material of an individual cell of unculturable bacteria. In PLoS One this week, Jan-Fang Cheng’s and Nancy Moran’s groups at JGI and the University of Arizona report the completed sequence of Candidatus Sulcia meulleri, obtained from an uncultured single cell. The Bacteroidetes Sulcia is one of two obligate bacterial symbionts inhabiting sharpshooters. A single Sulcia cell was sampled from the host bacteriome using an inverted microscope (Zeiss) and a micromanipulator, its genome amplified via multiple displacement amplification and sequenced using a combination of Sanger sequence and pyrosequencing, generating a total of 57 Mb of sequence. This approach can now be used to generate complete reference genomes urgently needed for metagenomic of bacterial communities.

Metagenomics of windshield splatters

November 30th, 2009

weld-splatter-park-2Metagenomics involves sampling and sequencing the genome sequences of a community of organisms inhabiting a common environment, ocean, soil, or human gut. In their paper “Windshield splatter analysis with the Galaxy metagenomic pipeline” Kosakovsky Pond et al. applied metagenomic methodologies to directly determine the taxonomic composition of bugs collected by the front bumper and windshield of a 2006 Dodge Caravan. As every driver knows, the windshield of a moving vehicle is subjected to numerous insect strikes and this study shows that it can be used as a collection device for representative sampling of eukaryotes from our surrounding environment. The 454 reads were analyzed using a comprehensive pipeline – the Galaxy platform – for phylogenetic profiling of metagenomic samples that includes all steps from processing and quality control of data generated by next-generation sequencing technologies to statistical analyses and data visualization (http://galaxyproject.org). The number of sequencing reads has been used as a proxy for the relative abundance of taxa and therefore used to contrast biodiversity estimates between geographic locations. Most reads from two trips in in the northeastern United States  map to bacterial species. Aside from green plants and a few animal roadkills, two insect groups, Diptera and Hemiptera, represented the majority of eukaryotic reads in both samples. This study demonstrates that 454 sequencing technology can identify eukaryotic taxa from random reads generated from environmental samples and it is it possible to assess species abundance between geographic locations.

Kosakovsky Pond S, Wadhawan S, Chiaromonte F, Ananda G, Wen-Yu Chung, Taylor J, Nekrutenko A and The Galaxy Team (2009) Windshield splatter analysis with the Galaxy metagenomic pipeline. Genome Research, http://www.genome.org/cgi/doi/10.1101/gr.094508.109.

Sequencing a tree-killing fungus by 454 and Illumina

September 29th, 2009

ponderosae

The tree-killing fungus Grosmannia clavigera (= Ophiostoma clavigerum) carried by the mountain pine beetle (MPB), Dendroctonus ponderosae, is devastating pine forests in British Columbia (Canada).  This sap-stain ascomycetous fungus grows rapidly in the host tree phloem and through the sapwood where it produces melanin that discolours the wood, and blocks the host tree’s water transport system. More than 15 million hectares of pine forests are currently affected by MPB. The pathogenic fungus recently crossed the Rocky Mountains into Alberta, raising the concern that pine forests across Canada may become affected. Bark beetles, beetle-associated tree-infecting fungi, and pine trees are three of the major interacting biological components of this epidemic. Genomics offers new approaches to delineate some of these complex biological interactions (see the TRIA project web site for details). Scott DiGuistini and his collaborators have combined combined conventional, 40-kb fosmid paired-end Sanger reads from an ABI 3730xl sequencer, single-end 454 reads from Roche GS20 and GS FLX sequencers, and paired-end reads from an Illumina Genome Analyzer sequencer for generating the  ~32.5 Mb draft genome sequence of G. clavigera. They developed a hybrid approach that uses the Forge and Velvet assemblers for generating the de novo draft genome sequences. This G. clavigera genome sequencing, together with the sequencing of the methylotrophic yeast Pichia pastoris, demonstrate that the new sequencing technologies can efficiently be used for generating genome draft for eukaryotic fungal genomes.

Scott DiGuistini et al. (2009) De novo genome sequence assembly of a filamentous fungus using Sanger, 454 and Illumina sequence data. Genome Biology 10: R94

Unexpected high fungal diversity in forest soils

August 28th, 2009

hetraieSoil fungi play a major role in ecological and biogeochemical processes in forest ecosystems. Little is known, however, about the structure and richness of different fungal communities and the distribution of functional ecological groups (pathogens, saprobes and symbionts). Within the framework of our metagenomics project aimed to assess the microbial diversity in temperate forests, we surveyed the fungal diversity in six different forest soils at the Breuil-Chenue long-term observatory using tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS-1). The paper reporting this study is now online on the New Phytologist Early View pages.

No less than 166 350 ITS reads were obtained from all samples. In each forest soil sample (4 g), approximately 30 000 reads were recovered, corresponding to around 1000 molecular operational taxonomic units (MOTU). Most MOTUs (81%) belonged to the Dikarya subkingdom (Ascomycota and Basidiomycota). Richness, abundance and taxonomic analyses identified the Agaricomycetes as the dominant fungal class. The ITS-1 sequences (73%) analysed corresponded to only 26 taxa. The most abundant MOTUs showed the highest sequence similarity to Ceratobasidium sp., Cryptococcus podzolicus, Lactarius sp. and Scleroderma sp.

This study, together with two other surveys exploring the diversity of fungal communities in  Quercus macrocarpa phyllosphere (Jumpponen & Jones, 2009) and the diversity of arbuscular mycorrhiza fungi in a boreonemoral forest (Öpik et al., 2009), validates the effectiveness of high-throughput 454 sequencing technology for the survey of soil fungal diversity. The large proportion of unidentified sequences, however, calls for curated sequence databases. The use of pyrosequencing on soil samples will likely accelerate the study of the spatiotemporal dynamics of fungal communities in forest ecosystems.

Buée et al. (2009) 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytologist, doi: 10.1111/j.1469-8137.2009.03003.x

Jumpponen & Jones (2009) Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Quercus macrocarpa phyllosphere. New Phytologist, doi: 10.1111/j.1469-8137.2009.02990.x

Öpik et al. (2009) Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest. New Phytologist, doi: 10.1111/j.1469-8137.2009.02920.x

Unwrapping the genome of a protein µfactory

August 25th, 2009

PicpaThe methylotrophic yeast Pichia pastoris has become a highly successful system for the expression of heterologous genes. It is widely used for the production of recombinant proteins. Several factors have contributed to its rapid acceptance, the most important of which include (1) A promoter derived from the alcohol oxidase I (AOX1) gene of P. pastoris that is uniquely suited for the controlled expression of foreign genes, (2) The strong preference of P. pastoris for respiratory growth, a physiological feature that greatly facilitates its culturing at high cell densities relative to fermentative yeasts and (3) strains capable of human-type N-glycosylation are available increasing the utility of this ‘humanized’ yeast for biopharmaceutical production. In addition, P. pastoris is a widely used model organism for studying peroxisomal biogenesis and methanol assimilation.

In the June issue of Nature Biotechnology, Nico Callewaert’s group (from Ghent VIB) published the genome sequence of P. pastoris. To my knowledge, this  is the first fungal genome published that has been assembled exclusively from ‘454 GS-FLX’ reads. Using this approach, they highly oversampled the genome (897,000, 20 times coverage) and generated 70,500 paired-end sequence tags, to enable the assembly of all but seven contigs into nine ‘supercontigs’ (plus the mitochondrial genome). The genome is organized in four chromosomes with a total estimated size of 9.4 Mbp and 5,313 protein-coding genes. To facilitate the ‘customization’ of novel strains for protein production, in-depth gene curations of genes involved in protein secretion, protein glycosylation and protein degradation were performed. The wealth of information generated by these genome sequence and annotation will likely facilitate the design of highly productive biopharmaceutical strains.

De Schutter, K., Lin, Y.-C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., Rouzé, P., Van de Peer, Y., Callewaert, L. (2009) Genome sequence of the recombinant protein production host Pichia pastoris, a methylotrophic yeast. Nature Biotechnology 27, 561 – 566.