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[2013.10.29] albertsen genomics metagenomics

This document provides an overview of genomics and metagenomics. It begins with an introduction to genomics, describing genome assembly, validation, and metabolic reconstruction. It then covers metagenomics, discussing its history, pitfalls, and potentials. Key points include that genomics analyzes the parts list of a single genome, while metagenomics analyzes the collective genomes of an entire microbial community. Metagenomics has been used to explore novel sequences from various environments, perform comparative analyses between ecosystems, and extract genomes from low-abundance species.

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Genomics and Metagenomics Mads Albertsen Introduction to community systems microbiology 2013 CENTER FOR MICROBIAL COMMUNITIES
Agenda Genomics • • • • Introduction Assembly Validation Metabolic reconstruction (SM @ Thursday) Metagenomics • History • Pitfalls • Potentials CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction Genome = Parts list of a single genome CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction How to get from sequenced DNA to metabolic model? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction Wet lab work Shear DNA Extract DNA Sequence Bioinformatics N Reads 50-500 bp Assembly Contigs 1kb – 100 kbp Scaffolding N Scaffolds Hopefully Mbp CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Definitions > 1 kbp insert A sequenced piece of DNA Paired-end read Sequencing both ends of a short DNA fragment Mate-pair read Sequencing both ends of a long DNA fragment The length of the DNA fragment Contig 300-600 bp insert Read Insert size 50-500 bp A set of overlapping DNA segments that represents a consensus region of DNA Scaffold Contigs separated by gaps of known length Coverage The number of times a specific position in the genome is covered by reads length N 4x CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Genome Fragment Sequence Paired-end reads Assemble Contig 1 Contig 10 Scaffold 1 Inspiration: http://goo.gl/VOZVVg Contig 19 Scaffold 2 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Sequencing Genome (3.000.000 letters) Inspiration: http://goo.gl/VOZVVg Assembly Reads (50-500 letters each) Genome (3.000.000 letters) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly “It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity,.... “ Dickens, Charles. A Tale of Two Cities. 1859. London: Chapman Hall Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Way too much data to make all vs. all comparison Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Step 1: Convert reads into kmers Reads theageofwi sthebestof astheageof worstoftim Imesitwast the hea eag age geo eof ofw fwi sth the heb ebe bes est sto tof ast sth the hea eag age geo eof wor ors rst sto tof oft fti tim ime mes esi sit itw twa was ast Kmers (k = 3) Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Step 2: Join kmers with n-1 overlap ast sth Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 the hea eag age geo eof CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Step 2: Join kmers with n-1 overlap ast eag eag age age geo geo eof eof ofw ebe bes est sto sto tof tof wor ast hea hea heb sth sth the the the ors rst was fwi oft fti twa itw sit esi mes ime tim Do the same for all reads… Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Step 3: Simplify the graph Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Contigs It was the = ≠ incredulity age be st epoch times wor wisdom of foolishness belief “It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity,.... “ Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 Dickens, Charles. A Tale of Two Cities. 1859. London: Chapman Hall CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly What is the minimum kmer size that results in a single contig? Kmer = 3 Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly What is the minimum kmer size that results in a single contig? Kmer = 3 Kmer = 10 Itwasthebestoftimesitwastheworstoftimesitwastheageofwisdomitwastheageoffoolishnessitwastheepochofbeliefitwastheepochofincredulity Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Repeat = repeated DNA sequence that can’t be spanned by reads Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly Why not just increase the kmer size? Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assembly theageofwi Kmer = 3 the hea eag age geo eof ofw fwi Kmer = 9 theageofw heageofwi Kmers with errors = 2/2 Errors! Kmers with errors = 3/8 Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation I’ve assembled my 4.3 Mbp genome into 25 scaffolds with a N50 of 553 kbp. Is it a good assembly? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation N50 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation Estimating repeat content CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation 4 repeats in 2 copies each CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation How could I close this genome? 4 repeats in 2 copies each CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation How complete is the genome? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation Survey of essential single copy genes across sequenced phyla Genes 100-106 Essential single copy genes (can also be used to identify contamination) Phyla CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation Inspect the assembly CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Validation • N50 does not make much sense • Repeat content versus the number of scaffolds • Calculate the percentage of essential genes • Inspect the assembly CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metabolic reconstruction 4.3 Mbp genome … and so what? (@Thursday) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics Mads Albertsen Introduction to community systems microbiology 2013 CENTER FOR MICROBIAL COMMUNITIES
Introduction Genome = Parts list of a single genome CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction Photo: D. Kunkel; color, E. Latypova Metagenome = Parts list of the community CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction ”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.” - J. Handelsman et al., 1998 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction ”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.” - J. Handelsman et al., 1998 PubMed: metagenom*[Title/Abstract] CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction ”...functional analysis of the collective genomes of soil microflora, which we term the metagenome of the soil.” - J. Handelsman et al., 1998 Sequencing costs PubMed: metagenom*[Title/Abstract] http://www.genome.gov/sequencingcosts/ CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Introduction Metagenomics ≠ Amplicon sequencing CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sequencing and assembly ≈3.000.000 bp pr. genome 150 bp reads ≈1000 bp+ contigs CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Assigning information Function Contigs Databases Binning Taxonomy CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What have metagenomics been used for? Exploration Rusch et al., 2007 Plos Biology • 6.3 Gbp of sequence (2x Human genomes, 2000 x Bacterial genomes) • Most sequences were novel compared to the databases Qin et al., 2010 Nature • • • • 127 Human gut metagenomes 600 Gbp sequence (200 x Human genomes) 3.3 million genes identified Minimal gut metagenome definded CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What have metagenomics been used for? Comparative Dinsdale et al., 2008 Nature • A characteristic microbial fingerprint for each of the nine different ecosystem types Specific functions Hess et al., 2011 Science • Identified 27.755 putative carbohydrate-active genes from a cow rumen metagenome • Expressed 90 candidates of which 57% had enzymatic activity against cellulosic substrates CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What have metagenomics been used for? Extracting genomes Garcia Martin et al., 2006 Nat. Biotechnol. Albertsen et al., 2013 Nat. Biotechnol. • Genome extraction from low complexity metagenome • Candidatus Accumulibacter phosphatis • The first genome of a polyphosphate accumulating organism (PAO) with a major role en enhanced biological phosphorus removal • Genome extraction of low abundant species (< 0.1%) from metagenomes • First complete TM7 genome • Access to genomes of the ”uncultured majority” CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Pitfalls CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics made easy Great resources – but use with care CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
MG-RAST example Contigs CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Dataset overview CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Taxonomy and Function overview Taxonomy Function CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Compare with other samples Samples Functional categories CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Pitfalls You always get billions of data! CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Pitfalls Is your DNA extraction OK? ... and the samples you want to compare with? Did you sequence enough? Did you know the GC bias of your protocol? Did you normalize for sequencing depth? Did you use the same sequencing platform? Assembly = data not quantitative! Are you comparing assembled data with reads? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Databases Contigs Databases Annotated metagenome ...you only see what is in the database CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
What is in the databases? Finshed Genomes in IMG Vs. Greengenes 16S rRNA database Genomes 16S Phyla 29 90 Class 46 249 Order 100 405 Species 1268 99322* *97% clustering Note: only including 1 strain pr. species CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
MG-RAST example Contigs 650.000 EBPR proteins with taxonomy assigned How similar are they to the genomes in the database? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sludge microbes vs. Database genomes 650.000 EBPR proteins Note: not abundance weighted CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sludge microbes vs. Database genomes 650.000 EBPR proteins 1.260.000 Human gut Qin et al., 2010 Nature RAST ID: 4448044.3 Note: not abundance weighted CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Sludge microbes vs. Database genomes The 7 genera with most EBPR proteins assigned CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes What is the effect of not having closely related genomes in the database? 1. Remove a genome from the database 2. Search the removed genome against the database CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Accumulibacter phosphatis blastp 4326 proteins Best hit Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Accumulibacter phosphatis blastp Azoarcus 4326 proteins Best hit Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Accumulibacter phosphatis blastp 4326 proteins MEGAN LCA Lowest common ancester (LCA) approach: Hit 1: Beta-proteobacteria 80% ID Hit 2: Gamma-proteobacteria 79% ID Hit 3: Actinobacteria 59% ID Assigned to Proteobacteria Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Accumulibacter phosphatis blastp 4326 proteins MEGAN LCA Lowest common ancester (LCA) approach: Hit 1: Beta-proteobacteria 80% ID Hit 2: Gamma-proteobacteria 79% ID Hit 3: Actinobacteria 59% ID Bacteria 325 Beta- 853 Genus 4326 proteins: • 27% correctly classified on genus level • 54% not assigned the correct class • 101 genera identified Rhodocyclaceae 1149 Assigned to Proteobacteria Proteobacteria 860 Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 No hits 261 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Phylum Nitrospira defluvii blastp 4268 proteins: • 1% correctly classified on phylum level MEGAN LCA Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Nitrospira defluvii blastp MEGAN LCA + KEGG What about function? Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Nitrospira defluvii blastp MEGAN LCA + KEGG Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Effect of missing genomes Nitrospira defluvii blastp MEGAN LCA + KEGG Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Implication of missing genomes Function A Function B Function C Function D CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Pitfalls You always get billions of data! CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics ”If you want to understand the ecosystem you need to understand the individual species in the ecosystem” CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics Lion + Eagle ≠ Flying Lion CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Potentials CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Who - when, where and why? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes? Culturing Few microorganisms can be easily cultured (<<5%) Microorganisms needs to be studied in their environment CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes? What you think you study What you actually study CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes? Culturing Few microorganisms can be easily cultured (<<5%) Microorganisms needs to be studied in their environment Single cell genomics Only routinely performed in specialized labs Very incomplete genomes (mean 40%, range 10-90%) https://www.bigelow.org/ CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
How do we get the genomes? Culturing Few microorganisms can be easily cultured (<<5%) Microorganisms needs to be studied in their environment Single cell genomics Only routinely performed in specialized labs Very incomplete genomes (mean 40%, range 10-90%) https://www.bigelow.org/ Metagenomics CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics Reads DNA extraction Sequencing 100++ Abundant species (≈3 Mbp each) 100-150 bp Assembly Why not full genomes? Contigs 1000+ bp CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics Reads DNA extraction Sequencing 100++ Abundant species (≈3 Mbp each) 100-150 bp Assembly Why not full genomes? Contigs 1000+ bp 1. Micro-diversity 2. Separation of genomes (Binning) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Extracting genomes Not 1 strain AAAAAAAAAAAAAA AAAAAAAAATAAAA AAAAAAAAACAAAA What you get TAAAA Assembly AAAAAAAAA AAAAA CAAAA Many closely related strains CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Extracting genomes High micro-diversity Low micro-diversity Short term enrichment CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Metagenomics Reads DNA extraction Sequencing 100++ Abundant species (≈3 Mbp each) 100-150 bp Assembly Why not full genomes? Contigs 1000+ bp 1. Micro-diversity 2. Separation of genomes (Binning) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning PhD student Genomic signatures: - GC / Codon usage - Tetranucleotide frequency + statistical method ”Binning” Complex sample CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning PhD student Genomic signatures: - GC / Codon usage - Tetranucleotide frequency + statistical method ”Binning” Complex sample Problems: - Short pieces of sequence (1-10kbp) - Local sequence divergence CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning Abundance Abundance Sequence composition-independent binning Sample 1 Sample 2 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning Abundance Abundance Sequence composition-independent binning Sample 2 Abundance Sample 2 Sample 1 Abundance Sample 1 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning 1. Reduce micro-diversity Abundance Sample 2 2. Use multiple related samples Abundance Sample 1 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning 1. Reduce micro-diversity Abundance Sample 2 2. Use multiple related samples Abundance Sample 2 Abundance Sample 1 Abundance Sample 1 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Binning • Nitrospira enrichment running for years • 3 dominant species • No micro-diversity H. Daims & C. Dorninger, DOME, University of Vienna CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
SBR reactor Full-scale EBPR plant Short term enrichment Days Albertsen et al., 2013 Nat. Biotech. 1. Reduction of (micro)-diversity CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
SBR reactor Full-scale EBPR plant Short term enrichment 2. Two different DNA extraction methods Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Colored using a set of 100 phylogenetic marker genes Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Colored using a set of 100 phylogenetic marker genes TM7-1 (1.6%) TM7-2 (0.7%) TM7-3 (0.2%) TM7-4 (0.06%) Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Colored using a set of 100 phylogenetic marker genes Zoom on target TM7-2 (0.7%) Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Colored using a set of 100 phylogenetic marker genes Zoom on target PCA on genomic signatures PC2 TM7-2 (0.7%) TM7-2 PC1 Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Colored using a set of 100 phylogenetic marker genes Candidatus Saccharimonas aalborgensis TM7-1 (1.6%) Candidate phylum TM7 Saccharibacteria Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Genome validation Assembly inspection Essential single copy genes Genes (HMM models) Phyla Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Multi-metagenome http://madsalbertsen.github.io/multi-metagenome/ Short: goo.gl/0ctA3 • • • • • Albertsen et al., 2013 Nat. Biotech. Guides Workflow scripts Example data All the code Reccomendations CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Complex samples ...add more samples! S. M. Karst, AAU CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
It’s just a potential! ..and a poorly translated description of it. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Understanding ecosystems Metabolites Meta-bolomics Proteins Extraction mRNA Meta-proteomics Meta-transcriptomics DNA In Situ methods Community structure Microbial functions Meta-genomics Microbial needs P-Removal: N-Removal: -Removal: Foaming: Ethanol production: CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Questions? ma@bio.aau.dk @MadsAlbertsen85 MadsAlbertsen Per H. Nielsen Simon J. McIllroy Søren M. Karst EB group University of Queensland C. Dorringer H. Daims M. Wagner University of Vienna G.W. Tyson P. Hugenholtz CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

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[2013.10.29] albertsen genomics metagenomics

  • 1.
    Genomics and Metagenomics Mads Albertsen Introduction tocommunity systems microbiology 2013 CENTER FOR MICROBIAL COMMUNITIES
  • 2.
    Agenda Genomics • • • • Introduction Assembly Validation Metabolic reconstruction (SM@ Thursday) Metagenomics • History • Pitfalls • Potentials CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 3.
    Introduction Genome = Partslist of a single genome CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 4.
    Introduction How to getfrom sequenced DNA to metabolic model? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 5.
    Introduction Wet lab work ShearDNA Extract DNA Sequence Bioinformatics N Reads 50-500 bp Assembly Contigs 1kb – 100 kbp Scaffolding N Scaffolds Hopefully Mbp CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 6.
    Definitions > 1 kbpinsert A sequenced piece of DNA Paired-end read Sequencing both ends of a short DNA fragment Mate-pair read Sequencing both ends of a long DNA fragment The length of the DNA fragment Contig 300-600 bp insert Read Insert size 50-500 bp A set of overlapping DNA segments that represents a consensus region of DNA Scaffold Contigs separated by gaps of known length Coverage The number of times a specific position in the genome is covered by reads length N 4x CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 7.
    Assembly Genome Fragment Sequence Paired-end reads Assemble Contig 1 Contig10 Scaffold 1 Inspiration: http://goo.gl/VOZVVg Contig 19 Scaffold 2 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 8.
    Assembly Sequencing Genome (3.000.000 letters) Inspiration: http://goo.gl/VOZVVg Assembly Reads (50-500letters each) Genome (3.000.000 letters) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 9.
    Assembly “It was thebest of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity,.... “ Dickens, Charles. A Tale of Two Cities. 1859. London: Chapman Hall Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 10.
    Assembly Way too muchdata to make all vs. all comparison Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 11.
    Assembly Step 1: Convertreads into kmers Reads theageofwi sthebestof astheageof worstoftim Imesitwast the hea eag age geo eof ofw fwi sth the heb ebe bes est sto tof ast sth the hea eag age geo eof wor ors rst sto tof oft fti tim ime mes esi sit itw twa was ast Kmers (k = 3) Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 12.
    Assembly Step 2: Joinkmers with n-1 overlap ast sth Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 the hea eag age geo eof CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 13.
    Assembly Step 2: Joinkmers with n-1 overlap ast eag eag age age geo geo eof eof ofw ebe bes est sto sto tof tof wor ast hea hea heb sth sth the the the ors rst was fwi oft fti twa itw sit esi mes ime tim Do the same for all reads… Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 14.
    Assembly Step 3: Simplifythe graph Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 15.
    Assembly Contigs It was the = ≠ incredulity age be st epoch times wor wisdom of foolishness belief “Itwas the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity,.... “ Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 Dickens, Charles. A Tale of Two Cities. 1859. London: Chapman Hall CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 16.
    Assembly What is theminimum kmer size that results in a single contig? Kmer = 3 Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 17.
    Assembly What is theminimum kmer size that results in a single contig? Kmer = 3 Kmer = 10 Itwasthebestoftimesitwastheworstoftimesitwastheageofwisdomitwastheageoffoolishnessitwastheepochofbeliefitwastheepochofincredulity Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 18.
    Assembly Repeat = repeatedDNA sequence that can’t be spanned by reads Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 19.
    Assembly Why not justincrease the kmer size? Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 20.
    Assembly theageofwi Kmer = 3 the hea eag age geo eof ofw fwi Kmer= 9 theageofw heageofwi Kmers with errors = 2/2 Errors! Kmers with errors = 3/8 Example: http://goo.gl/nMWDAk Velvet example courtesy of J. Leipzig 2010 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 21.
    Validation I’ve assembled my4.3 Mbp genome into 25 scaffolds with a N50 of 553 kbp. Is it a good assembly? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 22.
    Validation N50 CENTER FOR MICROBIALCOMMUNITIES | AALBORG UNIVERSITY
  • 23.
    Validation Estimating repeat content CENTERFOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 24.
    Validation 4 repeats in2 copies each CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 25.
    Validation How could Iclose this genome? 4 repeats in 2 copies each CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 26.
    Validation How complete isthe genome? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 27.
    Validation Survey of essentialsingle copy genes across sequenced phyla Genes 100-106 Essential single copy genes (can also be used to identify contamination) Phyla CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 28.
    Validation Inspect the assembly CENTERFOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 29.
    Validation • N50 doesnot make much sense • Repeat content versus the number of scaffolds • Calculate the percentage of essential genes • Inspect the assembly CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 30.
    Metabolic reconstruction 4.3 Mbpgenome … and so what? (@Thursday) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 31.
    Metagenomics Mads Albertsen Introduction tocommunity systems microbiology 2013 CENTER FOR MICROBIAL COMMUNITIES
  • 32.
    Introduction Genome = Partslist of a single genome CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 33.
    Introduction Photo: D. Kunkel;color, E. Latypova Metagenome = Parts list of the community CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 34.
    Introduction ”...functional analysis ofthe collective genomes of soil microflora, which we term the metagenome of the soil.” - J. Handelsman et al., 1998 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 35.
    Introduction ”...functional analysis ofthe collective genomes of soil microflora, which we term the metagenome of the soil.” - J. Handelsman et al., 1998 PubMed: metagenom*[Title/Abstract] CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 36.
    Introduction ”...functional analysis ofthe collective genomes of soil microflora, which we term the metagenome of the soil.” - J. Handelsman et al., 1998 Sequencing costs PubMed: metagenom*[Title/Abstract] http://www.genome.gov/sequencingcosts/ CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 37.
    Introduction Metagenomics ≠ Ampliconsequencing CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 38.
    Sequencing and assembly ≈3.000.000bp pr. genome 150 bp reads ≈1000 bp+ contigs CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 39.
  • 40.
    What have metagenomicsbeen used for? Exploration Rusch et al., 2007 Plos Biology • 6.3 Gbp of sequence (2x Human genomes, 2000 x Bacterial genomes) • Most sequences were novel compared to the databases Qin et al., 2010 Nature • • • • 127 Human gut metagenomes 600 Gbp sequence (200 x Human genomes) 3.3 million genes identified Minimal gut metagenome definded CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 41.
    What have metagenomicsbeen used for? Comparative Dinsdale et al., 2008 Nature • A characteristic microbial fingerprint for each of the nine different ecosystem types Specific functions Hess et al., 2011 Science • Identified 27.755 putative carbohydrate-active genes from a cow rumen metagenome • Expressed 90 candidates of which 57% had enzymatic activity against cellulosic substrates CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 42.
    What have metagenomicsbeen used for? Extracting genomes Garcia Martin et al., 2006 Nat. Biotechnol. Albertsen et al., 2013 Nat. Biotechnol. • Genome extraction from low complexity metagenome • Candidatus Accumulibacter phosphatis • The first genome of a polyphosphate accumulating organism (PAO) with a major role en enhanced biological phosphorus removal • Genome extraction of low abundant species (< 0.1%) from metagenomes • First complete TM7 genome • Access to genomes of the ”uncultured majority” CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 43.
    Pitfalls CENTER FOR MICROBIALCOMMUNITIES | AALBORG UNIVERSITY
  • 44.
    Metagenomics made easy Greatresources – but use with care CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 45.
    MG-RAST example Contigs CENTER FORMICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 46.
    Dataset overview CENTER FORMICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 47.
    Taxonomy and Functionoverview Taxonomy Function CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 48.
    Compare with othersamples Samples Functional categories CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 49.
    Pitfalls You always getbillions of data! CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 50.
    Pitfalls Is your DNAextraction OK? ... and the samples you want to compare with? Did you sequence enough? Did you know the GC bias of your protocol? Did you normalize for sequencing depth? Did you use the same sequencing platform? Assembly = data not quantitative! Are you comparing assembled data with reads? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 51.
    Databases Contigs Databases Annotated metagenome ...you onlysee what is in the database CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 52.
    What is inthe databases? Finshed Genomes in IMG Vs. Greengenes 16S rRNA database Genomes 16S Phyla 29 90 Class 46 249 Order 100 405 Species 1268 99322* *97% clustering Note: only including 1 strain pr. species CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 53.
    MG-RAST example Contigs 650.000 EBPRproteins with taxonomy assigned How similar are they to the genomes in the database? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 54.
    Sludge microbes vs.Database genomes 650.000 EBPR proteins Note: not abundance weighted CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 55.
    Sludge microbes vs.Database genomes 650.000 EBPR proteins 1.260.000 Human gut Qin et al., 2010 Nature RAST ID: 4448044.3 Note: not abundance weighted CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 56.
    Sludge microbes vs.Database genomes The 7 genera with most EBPR proteins assigned CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 57.
    Effect of missinggenomes What is the effect of not having closely related genomes in the database? 1. Remove a genome from the database 2. Search the removed genome against the database CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 58.
    Effect of missinggenomes Accumulibacter phosphatis blastp 4326 proteins Best hit Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 59.
    Effect of missinggenomes Accumulibacter phosphatis blastp Azoarcus 4326 proteins Best hit Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 60.
    Effect of missinggenomes Accumulibacter phosphatis blastp 4326 proteins MEGAN LCA Lowest common ancester (LCA) approach: Hit 1: Beta-proteobacteria 80% ID Hit 2: Gamma-proteobacteria 79% ID Hit 3: Actinobacteria 59% ID Assigned to Proteobacteria Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 61.
    Effect of missinggenomes Accumulibacter phosphatis blastp 4326 proteins MEGAN LCA Lowest common ancester (LCA) approach: Hit 1: Beta-proteobacteria 80% ID Hit 2: Gamma-proteobacteria 79% ID Hit 3: Actinobacteria 59% ID Bacteria 325 Beta- 853 Genus 4326 proteins: • 27% correctly classified on genus level • 54% not assigned the correct class • 101 genera identified Rhodocyclaceae 1149 Assigned to Proteobacteria Proteobacteria 860 Related genomes Bacteria 1268 Proteobacteria 564 Betaproteobacteria 84 Rhodocyclales 5 Rhodocyclaceae 5 No hits 261 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 62.
    Effect of missinggenomes Phylum Nitrospira defluvii blastp 4268 proteins: • 1% correctly classified on phylum level MEGAN LCA Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 63.
    Effect of missinggenomes Nitrospira defluvii blastp MEGAN LCA + KEGG What about function? Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 64.
    Effect of missinggenomes Nitrospira defluvii blastp MEGAN LCA + KEGG Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 65.
    Effect of missinggenomes Nitrospira defluvii blastp MEGAN LCA + KEGG Related genomes Bacteria Nitrospirae 1268 3 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 66.
    Implication of missinggenomes Function A Function B Function C Function D CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 67.
    Pitfalls You always getbillions of data! CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 68.
    Metagenomics ”If you wantto understand the ecosystem you need to understand the individual species in the ecosystem” CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 69.
    Metagenomics Lion + Eagle≠ Flying Lion CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 70.
    Potentials CENTER FOR MICROBIALCOMMUNITIES | AALBORG UNIVERSITY
  • 71.
    Who - when,where and why? CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 72.
    How do weget the genomes? Culturing Few microorganisms can be easily cultured (<<5%) Microorganisms needs to be studied in their environment CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 73.
    How do weget the genomes? What you think you study What you actually study CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 74.
    How do weget the genomes? Culturing Few microorganisms can be easily cultured (<<5%) Microorganisms needs to be studied in their environment Single cell genomics Only routinely performed in specialized labs Very incomplete genomes (mean 40%, range 10-90%) https://www.bigelow.org/ CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 75.
    How do weget the genomes? Culturing Few microorganisms can be easily cultured (<<5%) Microorganisms needs to be studied in their environment Single cell genomics Only routinely performed in specialized labs Very incomplete genomes (mean 40%, range 10-90%) https://www.bigelow.org/ Metagenomics CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 76.
    Metagenomics Reads DNA extraction Sequencing 100++ Abundant species(≈3 Mbp each) 100-150 bp Assembly Why not full genomes? Contigs 1000+ bp CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 77.
    Metagenomics Reads DNA extraction Sequencing 100++ Abundant species(≈3 Mbp each) 100-150 bp Assembly Why not full genomes? Contigs 1000+ bp 1. Micro-diversity 2. Separation of genomes (Binning) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 78.
    Extracting genomes Not 1strain AAAAAAAAAAAAAA AAAAAAAAATAAAA AAAAAAAAACAAAA What you get TAAAA Assembly AAAAAAAAA AAAAA CAAAA Many closely related strains CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 79.
    Extracting genomes High micro-diversity Lowmicro-diversity Short term enrichment CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 80.
    Metagenomics Reads DNA extraction Sequencing 100++ Abundant species(≈3 Mbp each) 100-150 bp Assembly Why not full genomes? Contigs 1000+ bp 1. Micro-diversity 2. Separation of genomes (Binning) CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 81.
    Binning PhD student Genomic signatures: -GC / Codon usage - Tetranucleotide frequency + statistical method ”Binning” Complex sample CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 82.
    Binning PhD student Genomic signatures: -GC / Codon usage - Tetranucleotide frequency + statistical method ”Binning” Complex sample Problems: - Short pieces of sequence (1-10kbp) - Local sequence divergence CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 83.
    Binning Abundance Abundance Sequence composition-independent binning Sample1 Sample 2 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 84.
    Binning Abundance Abundance Sequence composition-independent binning Sample2 Abundance Sample 2 Sample 1 Abundance Sample 1 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 85.
    Binning 1. Reduce micro-diversity AbundanceSample 2 2. Use multiple related samples Abundance Sample 1 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 86.
    Binning 1. Reduce micro-diversity AbundanceSample 2 2. Use multiple related samples Abundance Sample 2 Abundance Sample 1 Abundance Sample 1 CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 87.
    Binning • Nitrospira enrichment runningfor years • 3 dominant species • No micro-diversity H. Daims & C. Dorninger, DOME, University of Vienna CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 88.
    SBR reactor Full-scale EBPRplant Short term enrichment Days Albertsen et al., 2013 Nat. Biotech. 1. Reduction of (micro)-diversity CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 89.
    SBR reactor Full-scale EBPRplant Short term enrichment 2. Two different DNA extraction methods Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 90.
    Colored using aset of 100 phylogenetic marker genes Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 91.
    Colored using aset of 100 phylogenetic marker genes TM7-1 (1.6%) TM7-2 (0.7%) TM7-3 (0.2%) TM7-4 (0.06%) Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 92.
    Colored using aset of 100 phylogenetic marker genes Zoom on target TM7-2 (0.7%) Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 93.
    Colored using aset of 100 phylogenetic marker genes Zoom on target PCA on genomic signatures PC2 TM7-2 (0.7%) TM7-2 PC1 Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 94.
    Colored using aset of 100 phylogenetic marker genes Candidatus Saccharimonas aalborgensis TM7-1 (1.6%) Candidate phylum TM7 Saccharibacteria Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 95.
    Genome validation Assembly inspection Essentialsingle copy genes Genes (HMM models) Phyla Albertsen et al., 2013 Nat. Biotech. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 96.
    Multi-metagenome http://madsalbertsen.github.io/multi-metagenome/ Short: goo.gl/0ctA3 • • • • • Albertsen etal., 2013 Nat. Biotech. Guides Workflow scripts Example data All the code Reccomendations CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 97.
    Complex samples ...add moresamples! S. M. Karst, AAU CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 98.
    It’s just apotential! ..and a poorly translated description of it. CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 99.
    Understanding ecosystems Metabolites Meta-bolomics Proteins Extraction mRNA Meta-proteomics Meta-transcriptomics DNA In Situmethods Community structure Microbial functions Meta-genomics Microbial needs P-Removal: N-Removal: -Removal: Foaming: Ethanol production: CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
  • 100.
    Questions? ma@bio.aau.dk @MadsAlbertsen85 MadsAlbertsen Per H. Nielsen SimonJ. McIllroy Søren M. Karst EB group University of Queensland C. Dorringer H. Daims M. Wagner University of Vienna G.W. Tyson P. Hugenholtz CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY

Editor's Notes