Towards better tools for fungal environmental metagenomics


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2013 talk on progress towards better Fungal metagenomics tools

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Towards better tools for fungal environmental metagenomics

  1. 1. Towards better tools for fungal environmentalmetagenomicsJason StajichPlant Pathology & Microbiology twitter: hyphaltip, stajichlab, fungalgenomes
  2. 2. AcknowledgementsPeng  Liu Sapphire  Ear Univ  of  Colorado,  BoulderBrad  Cavinder Erum  Khan   Rob  Knight IIGB Computational CoreSofia  Robb Lorena  Rivera Daniel  McDonaldJinfeng  Chen Carlos  RojasAnastasia  Gio@ Megna  Tiwari Noah  Fierer Jessica  De  Anda Sco0  BatesSteven  Ahrendt Annie  Nguyen   Jon  LeffDivya  Sain   Ramy  WissaYizhou  Wang Marine  Biological  LaboratoryYi  Zhou Mitch  Sogin Sue  HuseRaghu  RamamurthyEdward  Liaw Argonne  Na@onal  LabGreg  Gu Folker  MeyerDaniel  Borcherding Henrik  Nilsson Keith  Seifert
  3. 3. Molecular Ecology of microbes• What microbes live where?• Using molecular techniques improve upon culture based methods reducing bias in just fast- growing and or culturable organisms.• Many efforts to examine Bacteria and Archaeal diversity with sequencing developed important standards - e.g. Human Microbiome Project.• Efforts towards improving methods of studying of fungi in the environment
  4. 4. Plantae Amoebozoa Choanozoa Metazoa Microsporidia Fungi Rozella Chytridiomycota BlastocladiomycotaMulticellular with Mucoromycotinadifferentiated tissues Entomophthoromycotina Zoopagomycotina Loss of flagellum Kickxellomycotina Glomeromycota Mitotic sporangia Pucciniomycotina Basidiomycota to mitotic conidia Ustilaginomycotina Regular septa Agaricomycotina Taphrinomycotina AscomycotaMeiotic sporangia to Saccharomycotinaexternal meiospores Pezizomycotina 1500 1000 500 0 Millions of years Stajich et al. Current Biol 2009
  5. 5. Fungi interact with many organisms 10.3389/fpls.2011.00100 Betsy Arnold doi: 10.3389/fpls.2011.00100Endophytes Mycorrhiza doi: 10.1016/j.pbi.2009.05.007, F. Martin
  6. 6. Organisms interacting with Fungi - fungi as the host REPORTS to the Midwest Regional Center of Excellence for was supported by the NIH Institutional NRSA T32 SOM Text Biodefense and Emerging Infectious Disease Research GM07067 to the Washington University School of Figs. S1 to S4 Plant + Fungus + Mycovirus (MRCE) and by NIH grant AI53298. The DDRCC is Medicine. Tables S1 and S2 supported by NIH grant DK52574. W.W.L. was supported References by the Clinical/Translational Fellowship Program of the Supporting Online Material MRCE, the W.M. Keck Foundation, and the NIH National 6 November 2006; accepted 14 December 2006 Research Service Award (NRSA) F32 AI069688-01. P.A.P. Materials and Methods 10.1126/science.1137195 A Virus in a Fungus in a Plant: S2). The 2.2-kb fragment (RNA 1) is involved in virus replication, as both of its ORFs are similar to viral replicases. The first, ORF1a, has 29% Three-Way Symbiosis Required for amino acid sequence identity with a putative RNA-dependent RNA polymerase (RdRp) from Thermal Tolerance the rabbit hemorrhagic disease virus. The amino acid sequence of the second, ORF1b, has 33% identity with the RdRp of a virus of the fungal Luis M. Márquez,1 Regina S. Redman,2,3 Russell J. Rodriguez,2,4 Marilyn J. Roossinck1* pathogen Discula destructiva. These two ORFs overlap and could be expressed as a single Downloaded from on September 18, 2012 A mutualistic association between a fungal endophyte and a tropical panic grass allows both DOI: 10.1126/science.1136237 organisms to grow at high soil temperatures. We characterized a virus from this fungus that is protein by frameshifting, a common expression strategy of viral replicases. The two ORFs of involved in the mutualistic interaction. Fungal isolates cured of the virus are unable to confer RNA 2 have no similarity to any protein with heat tolerance, but heat tolerance is restored after the virus is reintroduced. The virus-infected known function. As in most dsRNA mycovi- fungus confers heat tolerance not only to its native monocot host but also to a eudicot host, ruses, the 5′ ends (21 bp) of both RNAs are which suggests that the underlying mechanism involves pathways conserved between these two conserved. Virus particles purified from C. groups of plants. protuberata are similar to those of other fungal viruses: spherical and ~27 nm in diameter (fig. E ndophytic fungi commonly grow within known mutualistic endophyte, Epichloë festucae, S3). This virus is transmitted vertically in the plant tissues and can be mutualistic in but no phenotype has been associated with this conidiospores. We propose naming this virus some cases, as they allow plant adaptation virus (9). Curvularia thermal tolerance virus (CThTV) toAPPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2010, p. 4063–4075 Vol. 76, No. 12 to extreme environments (1). A plant-fungal Fungal virus genomes are commonly com- reflect its host of origin and its phenotype.0099-2240/10/$12.00 doi:10.1128/AEM.02928-09 symbiosis between a tropical panic grass from posed of double-stranded RNA (dsRNA) (10). The ability of the fungus to confer heatCopyright © 2010, American Society for Microbiology. All Rights Reserved. geothermal soils, Dichanthelium lanuginosum, Large molecules of dsRNA do not normally tolerance to its host plant is related to the and the fungus Curvularia protuberata allows occur in fungal cells and, therefore, their presence presence of CThTV. Wild-type isolates of C. both organisms to grow at high soil temperatures is a sign of a viral infection (9). Using a protocol protuberata contained the virus in high titers, as in Yellowstone National Park (YNP) (2). Field for nucleic acid extraction with enrichment for evidenced by their high concentration of dsRNA Diverse Bacteria Inhabit Living Hyphae of Phylogenetically and laboratory experiments have shown that when root zones are heated up to 65°C, non- symbiotic plants either become shriveled and dsRNA (11), we detected the presence of a virus in C. protuberata. The dsRNA banding pattern consists of two segments of about 2.2 and 1.8 kb. (~2 mg/g of lyophilized mycelium). However, an isolate obtained from sectoring (change in morphology) of a wild-type colony contained a Diverse Fungal Endophytesᰔ† chlorotic or simply die, whereas symbiotic plants tolerate and survive the heat regime. When A smaller segment, less than 1 kb in length, was variable in presence and size in the isolates very low titer of the virus, as indicated by a low concentration of dsRNA (~0.02 mg/g of lyophi- grown separately, neither the fungus nor the plant analyzed and, later, was confirmed to be a sub- lized mycelium). These two isolates were iden- Michele T. Hoffman and A. Elizabeth Arnold* alone is able to grow at temperatures above 38°C, genomic element, most likely a defective RNA tical by simple sequence repeat (SSR) analysis but symbiotically, they are able to tolerate ele- (fig. S1 and Fig. 1, A and B). Using tagged with two single-primer polymerase chain reac- Downloaded from Division of Plant Pathology and Microbiology, School of Plant Sciences, 1140 E. South Campus Drive, vated temperatures. In the absence of heat stress, random hexamer primers, we transcribed the tion (PCR) reactions and by sequence analysis of symbiotic plants have enhanced growth rate virus with reverse transcriptase (RT), followed by the rDNA ITS1-5.8S-ITS2 region (figs. S4 and University of Arizona, Tucson, Arizona 85721 compared with nonsymbiotic plants and also amplification and cloning. Sequence analysis S5). Desiccation and freezing-thawing cycles are show significant drought tolerance (3). revealed that each of the two RNA segments known to disrupt virus particles (12); thus, my- Received 3 December 2009/Accepted 20 April 2010 Fungal viruses or mycoviruses can modulate contains two open reading frames (ORFs) (fig. celium of the isolate obtained by sectoring was plant-fungal symbioses. The best known exam- ple of this is the hypovirus that attenuates the Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the virulence (hypovirulence) of the chestnut blight Fig. 1. Presence or absence of CThTV in different strains of C. interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently fungus, Cryphonectria parasitica (4). Virus regu- protuberata, detected by ethid- lation of hypovirulence has been demonstrated bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least experimentally in several other pathogenic fungi ium bromide staining (A), Northern blot using RNA 1 (B) one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylo- (5–8). However, the effect of mycoviruses on and RNA 2 (C) transcripts of Domestication: Ant farmed fungi mutualistic fungal endophytes is unknown. There genetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including repre- is only one report of a mycovirus from the well- the virus as probes, and RT- PCR using primers specific for sentatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photo- a section of the RNA 2 (D). The synthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria 1 Plant Biology Division, Samuel Roberts Noble Foundation, isolate of the fungus obtained Post Office Box 2180, Ardmore, OK 73402, USA. 2Depart- using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic ment of Botany, University of Washington, Seattle, WA by sectoring was made virus- free (VF) by freezing-thawing. 98195, USA. 3Department of Microbiology, Montana State Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions University, Bozeman, MT 59717, USA. 4U.S. Geological Sur- The virus was reintroduced into surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were vey, Seattle, WA 98115, USA. the virus-free isolate through hyphal anastomosis (An) with the wild type (Wt). The wild-type isolate of
  7. 7. How many species of Fungi are there? Mycol. Res. 9S (6): 641--655 (1991) Printed in Great Britain 641 1.5 Million based on fungus to plant ratio of 6:1 Presidential address 1990 The fungal dimension of biodiversity: magnitude, significance, and conservation D. L. HAWKSWORTH International Mycological Institute, Kew, Surrey TW9 3AF, UK American Journal of Botany 98(3): 426–438. 2011.Don’t forget the endophytes... Fungi, members of the kingdoms Chromista, Fungi S.str. and Protozoa studied by mycologists, have received scant consideration in discussions on biodiversity. The number of known species is about 69000, but that in the world is conservatively estimated at 15 million; six-times higher than hitherto suggested. The new world estimate is primarily based on vascular plant:fungus ratios in THE FUNGI: 1, 2, 3 … 5.1 MILLION SPECIES?1 and the soil... different regions. It is considered conservative as: (1) it is based on the lower estimates of world vascular plants; (2) no separate Meredith Blackwell2 provision is made for the vast numbers of insects now suggested to exist; (3) ratios are based on areas still not fully known mycologically; and (4) no allowance is made for higher ratios in tropical and polar regions. Evidence that numerous new species Department of Biological Sciences; Louisiana State University; Baton Rouge, Louisiana 70803 USA remain to be found is presented. This realization has major implications for systematic manpower, resources, and classification. Fungi have and continue to playa vital role in the evolution of terrestrial life (especially through mutualisms), ecosystem functionPremise of the study: Fungi are major decomposers in certain ecosystems and essential associates of many organisms. They • and the provide enzymes and drugs and serve as experimental organisms. In 1991, a landmark paper estimated that there are 1.5 million DOI:10.3732/ajb.1000298 maintenance of biodiversity, human progress, and the operation of Gaia. Conservation in situ and ex situ are complementary, andon the Earth. Because only 70 000 fungi had been described at that time, the estimate has been the impetus to search for fungi the significance of culture collections is stressed. International collaboration is required to develop a world inventory, quantify functional unknown fungi. Fungal habitats include soil, water, and organisms that may harbor large numbers of understudied previously roles, and for effective conservation. fungi, estimated to outnumber plants by at least 6 to 1. More recent estimates based on high-throughput sequencing methods Upwards of 6M species - Lee Taylor (pers suggest that as many as 5.1 million fungal species exist. • Methods: Technological advances make it possible to apply molecular methods to develop a stable classification and to dis- cover and identify fungal taxa. Biodiversity, the extent of biological variation on Earth, has species, or populations. Knowledge of all of these is pertinent • Key results: Molecular methods have dramatically increased our knowledge of Fungi in less than 20 years, revealing a mono- comm) come to the fore as a key issue in science and politics for the to a thorough appreciation of the fungal dimension, butkingdom and increased diversity among early-diverging lineages. Mycologists are making significant advances in phyletic here“Thus, the Fungi is likely equaled only by the Insecta with respect to eukaryote 1990s. First used as BioDiversity in the title of a scientific meeting in Washington, D.C. in 1986 (Wilson, 1988: p. v), it at other levels. species discovery, but many fungi remain to be discovered. I will centre on species biodiversity; that is basal to discussions • Conclusions: Fungi are essential to the survival of many groups of organisms with which they form associations. They also attract attention as predators of invertebrate animals, pathogens of potatoes and rice and humans and bats, killers of frogs and has been rapidly adopted as a contraction of biotic diversity crayfish, producers of secondary metabolites to lower cholesterol, and subjects of prize-winning research. Molecular tools in use and under development can be used to discover the world’s unknown fungi in less than 1000 years predicted at current new
  8. 8. Microbial Ecology is not just outside• Most humans spend majority of lives indoors• What are the organisms that live in the built environment?• Are there beneficial organisms that influence overlal composition of communities?• How does the composition change when environmental conditions change (moisture, temperature, food sources)
  9. 9. Microbial ecology Bik et al., 2012
  10. 10. Microbial Ecology in simple terms• Collecting what’s there (sampling and PCR amplifying) [LAB]• Put labels on things by matching to knowns (BLAST or other approach to see what matches in a database) [COMPUTER]• See what is different (compare communities) [COMPUTER]
  11. 11. Sampling and amplifying• Total DNA extracted from a sample - soil, plant tissue, swab• PCR with primers designed to amplify a conserved locus• Sequencing with Sanger sequencing -> Next Generation Sequencing
  12. 12. Metagenomics - Amplicon• Amplify a targeted locus for sequencing.• Works best if there are universal primers which can amplify from all the species of interest• For Bacteria most successful locus has been Ribosomal Small Subunit gene (16S) • Primers that work well to amplify most groups of Bacteria and Archea• Other loci are useful markers for sometimes better species resolution (phylogenetics) or community functional diversity by targeting a protein coding gene
  13. 13. Universal primers Development one of first primer sets andamplified regions of rRNA small subunit gene Woese, Pace
  14. 14. Barcoding for multiplexing samples
  15. 15. Fungal Markers for molecular ecology• Needs to be universally amplifying across all groups• Ribosomal rRNA ( • Small Subunit and Large Subunit genes • Internal Transcribed Spacer 1 and 2• Protein coding genes • EF1alpha, RPB1, RPB2 (Fungal Tree of Life project)
  16. 16. White, Bruns, Lee, Taylor mycolab/primers.htm
  17. 17. White, Bruns, Lee, Taylor mycolab/primers.htm
  18. 18. mycolab/primers.htm
  19. 19. There’s a data storm coming 320k curated Roche-454 1M sequences per run sequences Illumina HiSeq 2-3 Billion sequences per run (10-14 days) Illumina MiSeq 3-5 M reads (1 day) IonTorrent 4-8 M reads (2hrs)
  20. 20. Fungal-specific Challenges• Alignment of ITS• Establishment of a reference tree • Unalignable sequence into tree with LSU• Naming and Curation of datasets
  21. 21. The problem with Alignment of ITS ITS1 5.8S
  22. 22. ITS is most useful as a barcode sequence Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi Conrad L. Schocha,1, Keith A. Seifertb,1, Sabine Huhndorfc, Vincent Robertd, John L. Spougea, C. André Levesqueb, Wen Chenb, and Fungal Barcoding Consortiuma,2 a National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20892; bBiodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, ON, Canada K1A 0C6; cDepartment of Botany, The Field Museum, Chicago, IL 60605; and d Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre (CBS-KNAW), 3508 AD, Utrecht, The Netherlands Edited* by Daniel H. Janzen, University of Pennsylvania, Philadelphia, PA, and approved February 24, 2012 (received for review October 18, 2011) Six DNA regions were evaluated as potential DNA barcodes for the intron of the trnK gene. This system sets a precedent for Fungi, the second largest kingdom of eukaryotic life, by a multina- reconsidering CO1 as the default fungal barcode. tional, multilaboratory consortium. The region of the mitochondrial CO1 functions reasonably well as a barcode in some fungal cytochrome c oxidase subunit 1 used as the animal barcode was genera, such as Penicillium, with reliable primers and adequate excluded as a potential marker, because it is difficult to amplify in species resolution (67% in this young lineage) (9); however, fungi, often includes large introns, and can be insufficiently vari- results in the few other groups examined experimentally are in- able. Three subunits from the nuclear ribosomal RNA cistron were consistent, and cloning is often required (10). The degenerate compared together with regions of three representative protein- primers applicable to many Ascomycota (11) are difficult to as- coding genes (largest subunit of RNA polymerase II, second largest sess, because amplification failures may not reflect priming subunit of RNA polymerase II, and minichromosome maintenance mismatches. Extreme length variation occurs because of multiple protein). Although the protein-coding gene regions often had introns (9, 12–14), which are not consistently present in a species. MICROBIOLOGY a higher percent of correct identification compared with ribosomal Multiple copies of different lengths and variable sequences oc- markers, low PCR amplification and sequencing success eliminated cur, with identical sequences sometimes shared by several species them as candidates for a universal fungal barcode. Among the (11). Some fungal clades, such as Neocallimastigomycota (an regions of the ribosomal cistron, the internal transcribed spacer early diverging lineage of obligately anaerobic, zoosporic gut (ITS) region has the highest probability of successful identification fungi), lack mitochondria (15). Finally, because most fungi are for the broadest range of fungi, with the most clearly defined bar- microscopic and inconspicuous and many are unculturable, ro- code gap between inter- and intraspecific variation. The nuclear bust, universal primers must be available to detect a truly rep- ribosomal large subunit, a popular phylogenetic marker in certain resentative profile. This availability seems impossible with CO1. groups, had superior species resolution in some taxonomic groups, The nuclear rRNA cistron has been used for fungal dia- such as the early diverging lineages and the ascomycete yeasts, but gnostics and phylogenetics for more than 20 y (16), and its was otherwise slightly inferior to the ITS. The nuclear ribosomal components are most frequently discussed as alternatives to CO1 small subunit has poor species-level resolution in fungi. ITS will be (13, 17). The eukaryotic rRNA cistron consists of the 18S, 5.8S, formally proposed for adoption as the primary fungal barcode and 28S rRNA genes transcribed as a unit by RNA polymerase I. marker to the Consortium for the Barcode of Life, with the possibil- Posttranscriptional processes split the cistron, removing two in- ity that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. ternal transcribed spacers. These two spacers, including the 5.8S
  23. 23. Solutions• ITS is hard to align across diverse taxa, but LSU is not.• Marker with both sequences would be useful for both phylogenetic placement and barcoding. 5.8S LSU• ITS + LSU amplicon proposed - primer testing with Illumina is under testing - a bit too large by current chemistry but could work in the near future
  24. 24. Putting a name on it• Most sequences will not have identified names• Grouping all observed sequences together to define OTU clusters even if no name can be assigned• Curated ITS databases - UNITE project • ~300,000 sequences in UNITE, ~200,000 which are full length (SSU + ITS + LSU) • 50% are identified to a species level (18,000 distinct latin binomials)
  25. 25. UNITE project forH. Nilsson
  26. 26. Soil Clone Group 1 - highly abundant, uncultured organismPorter et al. 2008
  27. 27. Soil Clone Group 1 - highly abundant, uncultured organismPorter et al. 2008
  28. 28. What’s in a name? Would a mold by any other name smellas sweet?• “One fungus, one name” is eliminating dual nomeclature (naming of sexual and asexual forms separately)• How to name species from molecular data alone? PERSPEC Uncultured fungus clone unisequences#37-3808_2763 ITS2, PS • Name by close relatives on the tree? Uncultured fungus clone MOTU_2635_GVUGVSB04J56R4 18S rRNA gene, PS, ITS Uncultured fungus clone MOTU_3006_GVUGV5B04JIHT 18S rRNA gene Uncultured fungus clone MOTU_1888_GVUGV5B04JJTLJ 18S rRNA gene Uncultured fungus clone MOTU_2993_GOKCVYYY06HH12J 18S rRNA gene, PS, ITS Uncultured fungus clone MOTU_2930_GOKCVYYY06G7201 18S rRNA Fibulobasidium murrhardtense strain CB59109 18S rRNA gene Uncultured fungus clone MOTU_141_GOKCVYYY06G5FYL 18S rRNA gene, PS, ITS Uncultured Tremellales clone LTSP_EUKA_P4L03 18S rRNA gene, PS, ITS • Use marker loci that contain both ITS and LSU Uncultured fungus clone unisequence#65-3936_0554 ITS2, PS Uncultured fungus clone MOTU_601_GOK Uncultured basidiomycete ITS to better place sequence in tree. Fungi 3 leaves Uncultured fungus clone unise Uncultured Tremellales clone LTSP_EUKA Trichosporonales sp. LM559 18S rRNA gene Uncultured fungus clone unisequences #65-3574_00447, ITS2, PS Uncultured fungus clone MOTU_4349_GOKCVYYY06GR7WA 18S rRNA gene, PS, ITS2 Uncultured fungus clone unisequences#69-3466_2373 ITS2, PS • Proposal to name species in Botanical code Uncultured fungus clone MOTU_43 Uncultured fungus clone F66N0BQ02H1NX5 18S rRNA Uncultured fungus clone LT5P_EUKA_P5H04 18S rRNA gene, 18S–25/28S rRNA gene directly from sequence Uncultured fungus clone MOTU_1778_GVUGB5B04IF01X 18S rRNA gene, PS Uncultured fungus clone MOTU_4043_GVUGB5B04JK5N2 18S rRNA gene, PS, ITS2 Uncultured fungus clone MOTU_2412 Uncultured Agaricomycotina clone 6_g19 18S rRNA gene Uncultured fungus clone MOTU_3797_GOKCVYYY06HBZ1X 18S rRNA gene, PS, ITS2 Uncultured Rhodotorula IT51, 5.8S rRNA, ITS2 and partial 28S rRNA, clone MNIB2FAST_K1 Uncultured Tremellales clone 5_D20 18S rRNA, ITS1, 5.8S rRNA gene, ITS1• Good old fashioned microbiology Uncultured fungus clone U_QM_090130_127_1A_plate1g12.b1 18S rRNA gene, PS, ITS1 Uncultured fungus clone OTU_1445_1GW5CJXV07HXDTO 18S rRNA gene Uncultured fungus clone MOTU_3163_GYUGV5B0412KQP 18S rRNA gene, PS, ITS1 Uncultured fungus clone MOTU_533_GOKCVYYY06GU3JA18S rRNA gene, PS, ITS1 Uncultured fungus clone U_QM_090130_240_B_plate1a12.b1 18S rRNA gene, PS, ITS1 Uncultured fungus clone OTU_403_GW5CJXV07IOX5A 18S rRNA gene Uncultured fungus clone singleton_70-3063_2201 18S rRNA gene, PS, ITS HIbbett and Taylor 2013 gi|22497358|gb|FJ761130.1| uncultured fungus clone singleton_70-3063_2201 18S rRNA gene
  29. 29. From barcodes to organisms - low throughput but effective Dilution to Extinction (d2e)‘High throughput’ isolation from global dust samples Sarea resinae Cryptocoryneum rilstonei Keith Seifert
  30. 30. Communitycomparisons• Pie charts of taxonomic differences varied across treatments• 16S Community composition varies with smoking and COPD status Erb-Downward et al 2011.
  31. 31. Comparingcommunities• Taxonomic diversity varies across ant worker type and time of year
  32. 32. Workflows for data processing Bik et al., 2012
  33. 33. Tools - QIIME: Quantitative Insight Into Molecular Ecology• For amplicon based datasets (16s, 18s, ITS) • Alpha diversity - phylogenetic diversity, Chao, number of observed species • Generate species diversity plots to assess community diversity • Beta diversity - Unifrac distance, Bray-Curtis, Jaccard • Need reference phylogenetic tree to compute these, unavailable• Support for shotgun metagenomics
  34. 34. Approaches to clustering sequences• De novo clustering • Requires all-vs-all searches, very expensive• Known Knows - “Closed reference” • Match sequences to a database of representative known sequences • Fast, but throw out unknowns• Known Knowns and Known Unknowns - “Open reference” • Match to known set and de novo cluster the remainder
  35. 35. QIIME on fungal data• New (Dec 2012) Fungal ITS reference database from UNITE incorporated as QIIME resource• Can use it to match against known set (closed-reference) or match and cluster unknowns (open reference)• One dataset of Indoor dust samples from Kerry Kinney (UT Austin) group• A second indoor sampled (Amend et al)
  36. 36. QIIME taxonomic distribution for samples Greg Gu
  37. 37. A previously published indoor mycobiome• Amend et al PNAS 2010 “Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics.”• Sequencing dust from houses and office buildings• 72 samples of fungi from 6 continents. Sampled ITS2 region and the D1-D2 region of LSU with 454-FLX• A primary finding was increasing species diversity with increasing latitude
  38. 38. Fig 1. Amend et al 2010
  39. 39. MG-RAST with Fungal Data
  40. 40. MG-RAST summary statistics
  41. 41. Hits summarized by different taxonomic levels
  42. 42. Rarefaction curve (1 sample)
  43. 43. ITS 28SPCA  of  normalized  counts  –  Painted  by  rRNA  type MG-­‐RAST  tools
  44. 44. PCA  of  normalized  counts  –  Painted  by  sampled  country MG-­‐RAST  tools
  45. 45. PCA  of  normalized  counts  –  Painted  by  sampled  eleva@on MG-­‐RAST  tools
  46. 46. Metagenomics -shotgun approach• For non-amplicon based studies of community composition• Will be the future approaches for community studies with the increased sequencing depth• Metatranscriptomics for studying what is expressed• Support in QIIME and MG-RAST for the studies, but limited by the diversity of genome/protein sequences which can be matched.
  47. 47. Increasing fungal genome diversity
  48. 48. Fungal genome sequencing 400+ genomes of Fungi
  49. 49. Addressing the phylogenetic diversity: 1000 Fungal genomes !"#$%&%()*+#+,- !"#$%&%()*+#+,- .%#$/+%()*+#+,- .%#$/+%()*+#+,- 01"%2%()*+#+,- 01"%2%()*+#+,- D+%E8%,,%()*+#+,- D+%E8%,,%()*+#+,- F+GG%()*%2&4- 3&*+"#4+-,+/,- F+GG%()*%2&4- 3&*+"#4+-,+/,- 547%187+&%()*+#+,- 547%187+&%()*+#+,- 5+*4&%"%()*+#+,- 5+*4&%"%()*+#+,- 5+%2%()*+#+,- 5+%2%()*+#+,- 5*$&%()*+#+,- 5*$&%()*+#+,- 6"78%()*+#+,- 6"78%()*+#+,- F+GG%()*+#+,- F+GG%()*+#+,- H%"/4"%()*+#+,- H%"/4"%()*+#+,- H4**$4"%()*%2&4- H4**$4"%()*+#+,- H4**$4"%()*%2&4- H4**$4"%()*+#+,- I+%8+*#%()*+#+,- I+%8+*#%()*+#+,- J4K$"&%()*%2&4- F&+1(%*),2/%()*+#+,- J4K$"&%()*%2&4- F&+1(%*),2/%()*+#+,- H*$G%,4**$4"%()*+#+,- H*$G%,4**$4"%()*+#+,- J4K$"&%()*+#+,- J4K$"&%()*+#+,- M,284E&%()*%2&4- 0L%74,/%()*+#+,- M,284E&%()*%2&4- 0L%74,/%()*+#+,- M,284E&%()*+#+,- M,284E&%()*+#+,- !E4"*%,287%()*+#+,- !E4"*%,287%()*+#+,- !#"4*2+88%()*+#+,- !#"4*2+88%()*+#+,- N84,,*18%()*+#+,- N84,,*18%()*+#+,- F1**&%()*%2&4- N")K#%()*%*%84*%()*+#+,- F1**&%()*%2&4- N")K#%()*%*%84*%()*+#+,- N),#%74,/%()*+#+,- N),#%74,/%()*+#+,- O*"%7%#")%()*+#+,- O*"%7%#")%()*+#+,- OL%()*+#+,- OL%()*+#+,- F1**&%()*+#+,- F1**&%()*+#+,- !E4"*%()*+#+,- !E4"*%()*%2&4- !E4"*%()*%2&4- !E4"*%()*+#+,- .4*")()*+#+,- .4*")()*+#+,- J"+(+88%()*+#+,- J"+(+88%()*+#+,- D8%(+"%()*+#+,- D8%(+"%()*+#+,- 0&#%(%K$#$%"%()*%2&4- 0&#%(%K$#$%"%()*%2&4- P*Q+88%()*%2&4- O%"2+"+88%()*%2&4- P*Q+88%()*%2&4- O%"2+"+88%()*%2&4- 04"8)-.T+"E&E- O1*%"%()*%2&4- R%%K4E%()*%2&4- 04"8)-.T+"E&E- O1*%"%()*%2&4- 5&+4E+,- R%%K4E%()*%2&4- I+%*488(4,2E%()*+#+,- S84,#%*84/%()*+#+,- 5&+4E+,- I+%*488(4,2E%()*+#+,- N$)#"/%()*+#+,- S84,#%*84/%()*+#+,- O%&%78+K$4"/%()*+#+,- N$)#"/%()*+#+,- O%&%78+K$4"/%()*+#+,- 9- <9- >9- @9- B9- ;99- ;<9- 9:- ;9:- <9:- =9:- >9:- ?9:- @9:- A9:- B9:- C9:- ;99:- NumbersBlue = completed or in progress, Red= proposed for Tier One sampling, or Percent of Families in each clade and their current or proposed genomeGreen = remaining unsampled families sampling
  50. 50. Community can propose new genomes
  51. 51. Summary• Fungal microbial ecology is embracing highthroughput sequencing technologies for community studies• Limitations due to lack of curated sequences and the properties of the marker loci used• Building new databases and tools to help with the analyses will improve utility• Improvements in sequencing chemistry (read length x depth) make this a moving target for establishing the best practices• Deeper studies will improve our understanding of the fungal diversity and role of fungi in different ecosystems - 1000 genomes project can help provide anchor representatives of this diversity.
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