Gene and genome discovery throughmetagenomicsgene synthesis and single cell               genomics            Eddy Rubin  ...
Reduce our Dependence     onPETROLEUM• Decreased Consumption    (Improved Efficiency)• New Renewable Sources          (Bio...
Do We Need Biofuels and Why Not Just Batteries                              ?                   Energy Density  100 fold m...
Cellulosic Biofuels   CO2                                CO2Biomass            CO2           Land Use
Cellulosic Biofuel ProductionNeed to discover of enzymes of greater diversity                      Inefficient            ...
Biomass degradation communities     being studied by the JGIMarsupials         Birds            Insects                   ...
Cow Rumen Highly Efficient at  Biomass Deconstruction         Fistulated Cow
36 hr  Biomass                                         ~55% reduction in(Switchgrass)                                     ...
½ Tb of Rumen Sequence       Gb                                          This study300,000 600,000      Warnecke 07       ...
Total Assembly Based Pipeline                       Gene            HMMSearchAssembly             prediction       (Glyco ...
Genes Known to Deconstruct        Cellulose                                                            Cellulose          ...
Many in “computational” enzymes predictedBut are the in silica predictions real & functional?Digital Information          ...
From Sequence Datato Function Info       From Sequence to FunctionalSAMPLE                                 SEQUENCE EXPRES...
GH1 Functional Analysis                       Industrial process requires:                       -Activity at 70 degrees C...
Select 300 Candidates for Synthesis and Functional Characterization Maximizing Phylogenetic Space Cellobiose              ...
Synthetic GH1 activity profiles           (Temperature & pH ) Settemperature optima17 Enzymes identified active at pH 4.5 ...
How can we use this information  to improve bioenergy yield?
How can we use this information  to improve bioenergy yield?                        Biofuel Amylase                       ...
Conclusion Trawling deep metagenomic data is a successfulstrategy to massively add to the diversity of enzymes            ...
Conclusion Trawling deep metagenomic data is a successfulstrategy to massively add to the diversity of enzymes            ...
Information in Genomes vs GenesPathways, Which organism is doing what, Capabilities of                particular organism…
Short Read Assemblers             (varied parameters combined results)                              0.5 TB MetagenomeAssem...
What we have are scaffolds but no Genomes    Bin of Scaffolds into Draft Genomes  Tetra Nucleotide Frequency and Sequence ...
Assembled from Cow Rumen Microbiome 15 Draft Genomes   None of Which Have Ever Been Previously Reported                   ...
Proof that in silica assembled genomes of hard to culture        organisms without a reference genome?
Single Cell Genome Sequencing          72h                             rumen community                             FACS   ...
Metagenomic versus Single Cell                  Derived Genome                                       Binned MetagenomicSca...
ConclusionUltra deep metagenomicsequencing, even with shortreads, likely to increasinglybecome a method of choice toidenti...
Voxelation  Gene expression tomography           Alex Sczyrba                 Matthias Hess                 TanjaWoyke    ...
Q&A
Discovery of Cow Rumen Biomass-Degrading Genes and Genomes through DNA Sequencing, DNA Synthesis and Single Cell Genomics,...
Discovery of Cow Rumen Biomass-Degrading Genes and Genomes through DNA Sequencing, DNA Synthesis and Single Cell Genomics,...
Discovery of Cow Rumen Biomass-Degrading Genes and Genomes through DNA Sequencing, DNA Synthesis and Single Cell Genomics,...
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Discovery of Cow Rumen Biomass-Degrading Genes and Genomes through DNA Sequencing, DNA Synthesis and Single Cell Genomics, DOE Joint Genome Institute, Eddy Rubin, Copenhagenomics 2012

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Discovery of Cow Rumen Biomass-Degrading Genes and Genomes through DNA Sequencing, DNA Synthesis and Single Cell Genomics

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  • At JGI we looked quite a few organisms that live on plant materials. These includes herbivore mammals, birds, insects and even mollusks. The share one thing in common: none of these carry cellulolytic enzyme genes in their own genome, they rely on the microbial communities in their guts, for biomass degradation. Here we chose the cow to study the microbiome in its rumen, the reason why we did this is on the next slide.
  • Compared to two similar enzyme discovery studies from biomass degradation communities done previously at JGI, we obtained a massive amount of data. Four years ago Termite hindgut using Sanger sequencing got 71Mb. Last year From Wallaby foregut microbial communities, using a combination of Sanger and 454, and got about 800Mb of sequences. With more data we expected to find more enzymes encoded by the rumen microbes, but before everything we’ll have to deal with this terabyte scale data beast.
  • From this strategy we we assembled 15in silica draft genomesWith the single cell datata available we can now ask the question whether the draft genomes are real matching the single cell genomeThe question does this represent a real genomeThe first thing we see is that each scafolds have a significant number of hits from the single cell data which indicates that the set of scafolds in this draft genome bin are dervied from the same organsmThe second observation is that the majority of reads from the single cell genome maps to this draft genome suggesting that our draft does include most of the active genome pof this oranism this indicates
  • Discovery of Cow Rumen Biomass-Degrading Genes and Genomes through DNA Sequencing, DNA Synthesis and Single Cell Genomics, DOE Joint Genome Institute, Eddy Rubin, Copenhagenomics 2012

    1. 1. Gene and genome discovery throughmetagenomicsgene synthesis and single cell genomics Eddy Rubin Lawrence Berkeley National Lab Berkeley CA
    2. 2. Reduce our Dependence onPETROLEUM• Decreased Consumption (Improved Efficiency)• New Renewable Sources (Biofuels)
    3. 3. Do We Need Biofuels and Why Not Just Batteries ? Energy Density 100 fold more energy / kilo of petroleum than in a kilo of the very best battery• A 747 jet needs 200,000 kilos of fuel to fly from SF to Copenhagen. With a 20,000,000 kilo battery a 747 jet won’t get off the ground
    4. 4. Cellulosic Biofuels CO2 CO2Biomass CO2 Land Use
    5. 5. Cellulosic Biofuel ProductionNeed to discover of enzymes of greater diversity Inefficient with newproperties & Expensive Enzymes Biofuels Biomass Deconstruction Fermentation and Fuel Synthesis
    6. 6. Biomass degradation communities being studied by the JGIMarsupials Birds Insects Mollusks Hoatzin (Stink Bird) Ruminate
    7. 7. Cow Rumen Highly Efficient at Biomass Deconstruction Fistulated Cow
    8. 8. 36 hr Biomass ~55% reduction in(Switchgrass) cellulose Fistulated Cow IlluminaHiSeq 4 billion reads (200bp, 3kb, 5kb) Metagenomic Fiber-attached DNA ~ ½ Terabase of Sequence Data microbes
    9. 9. ½ Tb of Rumen Sequence Gb This study300,000 600,000 Warnecke 07 (Sanger, Termite Hindgut) 1/2 of a TB 500,000250,000200,000 400,000150,000 300,000 200,000100,000 71Mb 80Mb100,00050,000 0 Termite hindgut TammarWallaby
    10. 10. Total Assembly Based Pipeline Gene HMMSearchAssembly prediction (Glyco Hydrol & CB Module domains) 179,092 scaffolds Thousands of Potential 2.5 M genes Cellulolytic Enzymes
    11. 11. Genes Known to Deconstruct Cellulose Cellulose -(1-4) Endoglucanase EC 3.2.1.4 Cellobiohydrolase EC 3.2.1.91 & EC 3.2.1.150 -Glucosidase EC 3.2.1.21 & EC 3.2.1.74 Previously Newly Discovered in Known Total Rumen Data 3350 11 ~556
    12. 12. Many in “computational” enzymes predictedBut are the in silica predictions real & functional?Digital Information Functional Information (Sequence Data) ???????????? (Biochemical Activity) Large Scale Gene Synthesis
    13. 13. From Sequence Datato Function Info From Sequence to FunctionalSAMPLE SEQUENCE EXPRESSIONDATA ANALYSIS SYNTHESIS VALIDATE SYSTEM Next-Gen Expression Oligo synthesis and assembly Sequencing Vectors
    14. 14. GH1 Functional Analysis Industrial process requires: -Activity at 70 degrees CCellobiose -Stability at pH 4.5 GH1Glucose
    15. 15. Select 300 Candidates for Synthesis and Functional Characterization Maximizing Phylogenetic Space Cellobiose GH1 Glucose
    16. 16. Synthetic GH1 activity profiles (Temperature & pH ) Settemperature optima17 Enzymes identified active at pH 4.5 & 70 degrees C
    17. 17. How can we use this information to improve bioenergy yield?
    18. 18. How can we use this information to improve bioenergy yield? Biofuel Amylase Corn
    19. 19. Conclusion Trawling deep metagenomic data is a successfulstrategy to massively add to the diversity of enzymes with desired activities
    20. 20. Conclusion Trawling deep metagenomic data is a successfulstrategy to massively add to the diversity of enzymes with desired activitiesCan we assemble genomes from deep metagenomic data?
    21. 21. Information in Genomes vs GenesPathways, Which organism is doing what, Capabilities of particular organism…
    22. 22. Short Read Assemblers (varied parameters combined results) 0.5 TB MetagenomeAssembly 1 Assembly 2 Assembly 3 Clustering Contigs & Scaffolds
    23. 23. What we have are scaffolds but no Genomes Bin of Scaffolds into Draft Genomes Tetra Nucleotide Frequency and Sequence Coverage Fragments (scaffolds) of DNA from the same bacterial species have a similar tetranucleotideferquenciy Fibrobacteres Proteobacteria Cyanobacteria
    24. 24. Assembled from Cow Rumen Microbiome 15 Draft Genomes None of Which Have Ever Been Previously Reported (1.8-3.3 MB)
    25. 25. Proof that in silica assembled genomes of hard to culture organisms without a reference genome?
    26. 26. Single Cell Genome Sequencing 72h rumen community FACS single cellsMultiple Displacement Amplification (MDA) Shot gun sequencing and genome assembly of DNA from isolated single amplified genomes single cell
    27. 27. Metagenomic versus Single Cell Derived Genome Binned MetagenomicScaffolds (Draft In Silica 3.1 Mb Genome) Single Cell Genome Reads Single Cell Genome Reads Map to every MetagenomicScaffold Suggests that the scaffolds that bin together are from the same organism>90% of single cell genome reads map to this single draft genome Suggests that the draft genome is fairly complete
    28. 28. ConclusionUltra deep metagenomicsequencing, even with shortreads, likely to increasinglybecome a method of choice toidentify genes and characterizethe genomes of unculturedorganismsWill enable the exploitation of thediverse capabilities present inenvironmental organisms to offerbiotech solutions
    29. 29. Voxelation Gene expression tomography Alex Sczyrba Matthias Hess TanjaWoyke Voxelation + = GETLo thruput + Hi thruput = Hi thruput3D info 0D info 3D info Crump Institute for Molecular Imaging
    30. 30. Q&A

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