Day 3 Examine gels from PCR Learn about more molecular methods in microbial ecology Tour the Bay Paul Center Keck Sequencing Facility
1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control Genes We Targeted
1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control Emily S Angela C
1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control Allison T Nia B
1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control Jacob Kendall
1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control Jessie Melissa
1: dsrab 1800bp 2: mcra 750bp 3: Bacteria 1450bp 4: Archaea 950bp 5: Archaea + 950bp 6: Negative control Emily O Gabriela
Some Problems with PCR Inhibitors in template DNA Amplification bias Gene copy number Limited by primer design Differential denaturation efficiency Chimeric PCR products may form Contamination w/ non-target DNA Potentially low sensitivity and resolution General screw-ups
So you have a positive PCR product: Now what? Get community fingerprint via T-RFLP, DGGE, etc. Clone and sequence Quantify it Go straight into sequencing (next generation sequencing)
Microbial community Community sampling approach Amplify single gene, for example, gene encoding 16S rrna Sequence and generate tree Extract total community DNA DNA Environmental genomics approach Restriction digest total DNA and then shotgun sequence, OR sequence directly (without cloning) using a high throughput DNA sequencer Assembly and annotation Outcomes Partial genomes Single-gene phylogenetic tree 1. Phylogenetic snapshot of most members of the community 2. Identification of novel phylotypes Total gene pool of the community 1. Identification of all gene categories 2. Discovery of new genes 3. Linking of genes to phylotypes 2012 Pearson Education, Inc.
B. Crump
What do you DO with sequences? Perform a similarity search Align the sequences Build a tree and classify Reconstruct genomes Categorize functions Compare organisms/samples Design probes and quantify Examine expression patterns Etc. Etc. Etc.
BLAST Basic Local Alignment Search Tool http://blast.ncbi.nlm.nih.gov/blast.cgi
Making Sense of Sequences: Molecular Phylogeny 1. Align sequences so that homologous residues are juxtaposed. 2. Count the number of differences between pairs of sequences; this is some measure of evolutionary distance that separates the organisms. 3. Calculate the tree, the relatedness map, that most accurately represents all the pairwise differences.
Quantitative PCR
Found similar novel dsr sequences in the sulfate-rich and methane-rich zones Different (and already known) dsr sequences in SMTZ
Quantitative PCR
Quantitative (Real Time) PCR Real time PCR monitors the fluorescence emitted during the reactions as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection
Quantitative (Real Time) PCR Detection of amplification-associated fluorescence at each cycle during PCR No gel-based analysis Computer-based analysis Compare to internal standards Must insure specific binding of probes/dye
Quantitative PCR Used qpcr to quantify total bacteria (16S rrna) and total sulfate reducers (dsr)
Quantitative PCR Sulfate reducing bacteria (dsr gene) peaks at SMTZ
Moving from who is there to who is active DNA RNA Proteins www.nobelprize.org
Reverse Transcription PCR (RT-PCR) Looks at what genes are being expressed in the environment Isolate mrna Reverse transcribe mrna to produce complementary DNA (cdna) Amplify cdna by PCR Analyze genes from environment
RT-PCR RNA + Reverse Transcriptase + dntps= cdna cdna + Primers + Taq + dntps = gene of interest Who is active? What genes are active?
qrt-pcr Gene copy and transcript numbers are greatest at the estuary head (Hythe), where the rates of denitrification/dnra are highest.
All require a priori knowledge to design primers or probes Community sampling approach Amplify single gene, for example, gene encoding 16S rrna Sequence and generate tree Extract total community DNA DNA Microbial community Environmental genomics approach Restriction digest total DNA and then shotgun sequence, OR sequence directly (without cloning) using a high throughput DNA sequencer Assembly and annotation Outcomes Partial genomes Single-gene phylogenetic tree 1. Phylogenetic snapshot of most members of the community 2. Identification of novel phylotypes Total gene pool of the community 1. Identification of all gene categories 2. Discovery of new genes 3. Linking of genes to phylotypes 2012 Pearson Education, Inc.
Sequencing Revolution
http://www.ipc.nxgenomics.org
Traditional 16S rrna Cloning Schematic courtesy of B. Crump
NextGen Approaches Schematic courtesy of B. Crump
What is the difference between standard and next-gen sequencing?
A few factoids: In 1920, the term genome was proposed by the German botanist, Hans Winkler to denote the totality of all genes on all chromosomes in the nucleus of a cell. It was derived from GENe and chromosome. In late 60 s, first trna sequence (80 nt) required 4 yrs. 1972 5S rrna (119 bp) required ~1 year. - Sanger 2D finger printing (RNAseq based) 1982-16S rrna (1450 bp) required ~1 year Maxam-Gilbert (Chemical method). 1986-18S rrna (~1760 bp) required ~1 week (Sanger di-deoxynucleotide chain termination). 1996- DNA sequencers produce ~40,000 bp/day-labeled chain terminators. 2002- DNA sequencers produce ~100,000 bp/day-labeled chain terminators).
A few factoids: In 1920, the term genome was proposed by the German botanist, Hans Winkler to denote the totality of all genes on all chromosomes in the nucleus of a cell. It was derived from GENe and chromosome. In late 60 s, first trna sequence (80 nt) required 4 yrs. 1972 5S rrna (119 bp) required ~1 year. - Sanger 2D finger printing (RNAse based) 1982-16S rrna (1450 bp) required ~1 year Maxam-Gilbert (Chemical method). 1986-18S rrna (~1760 bp) required ~1 week (Sanger di-deoxynucleotide chain termination). 1996- DNA sequencers produce ~40,000 bp/day-labeled chain terminators. 2002- DNA sequencers produce ~100,000 bp/day-labeled chain terminators). 2004- DNA sequencers ( 454 technology) produce 20,000,000 bp/4 hours. 2007- DNA sequencers ( 454 technology) produce 100,000,000 bp/6 hours. 2015- DNA sequencers ( Illumina produce 120,000,000,000 bp/24 hours.
Metagenomics
Metagenomics
Metagenomics Access genomes of uncultured microbes: Functional Potential Metabolic Pathways Horizontal Gene Transfer
2012 Pearson Education, Inc. Reconstruct Genomes
2012 Pearson Education, Inc. Categorize Functions
Metagenomics 16s rrna gene Gammaproteobacteria SAR86 Proteorhodopsin gene
Proteorhodopsin gene
A new way of using sunlight in the surface ocean DeLong EF, Béjà O (2010) The Light-Driven Proton Pump Proteorhodopsin Enhances Bacterial Survival during Tough Times. PLoS Biol 8(4): e1000359. doi:10.1371/journal.pbio.1000359 http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000359
Proteorhodopsins occur in 13%-80% of marine bacteria and archaea in oceanic surface waters Venter et al. 2004
Moving from who is there to who is active DNA RNA Proteins www.nobelprize.org
Metatranscriptomics Access expressed genes of uncultured microbes Looking at expression of defined genes via PCR GeoChip-type analyses with RNA Etc.
Stable Isotope Probing (SIP) Links specific metabolic activity to diversity using a stable isotope Microorganisms metabolizing stable isotope (e.g., 13 C) incorporate it into their DNA DNA with 13 C can then be used to identify the organisms that metabolized the 13 C SIP of RNA also possible
Who are the active autotrophs of the subseafloor? What metabolic pathways are they using across geochemical gradients? How do viruses impact the subseafloor community? How does this new production influence deep carbon cycling?
Diverse Metabolisms
Venting fluids as window into subseafloor biosphere
RNA-SIP Vent Fluid
RNA Concentration (ng/ul) RNA-SIP 2.5 RNA Precipitation 2 1.5 1 12C-30 0.5 13C-30 0 1.77 1.78 1.79 1.8 1.81 1.82 1.83 1.84 1.85 1.86 Buoyant Density
RNA Concentration (ng/ul) Uptake of Bicarbonate 2.5 2 1.5 12L 13H Active autotrophs 1 12C-30 13C-30 0.5 13L 12H 0 1.77 1.78 1.79 1.8 1.81 1.82 1.83 1.84 1.85 1.86 Buoyant Density
30 C, 36 hr Marker 113 Vent Active autotrophs 55 C, 36 hr Active autotrophs 80 C, 18 hr Active autotrophs Fortunato & Huber, In Review
mrna Taxonomy Caminibacter Sulfurimonas Methanocaldococcus Nautilia Methanothermococcus Fortunato & Huber, In Review
Carbon Fixation Pathways Fortunato & Huber, In Review
Energy Fortunato & Huber, In Review
Not all Vents are the Same! Vent Characteristic: Anemone Marker 33 Marker 113 Temperature, C 28.5 27.8 24.1 Hydrogen sulfide, mmol L -1 1.1 0.6 0.6 Ammonia, µmol L -1 1.3 3.8 3.5 ph 5.5 5.5 6.2 Hydrogen, µmol kg -1 13.9 1.2 < 1.0 Methane, µmol kg -1 16.7 15.8 13.2 Oxygen, µmol L -1 13.0 12.0 7.0
mrna: 13 C, 80 C Aquifex Desulfurobacterium Sulfur, nitrate reduction Hydrogen oxidation Methanogenesis Carbon fixation Methanocaldococcus Thermovibrio Methanocaldococcus Anemone Mkr 33 Mkr 113 Fortunato et al, Unpub
mrna: 13 C, 80 C Aquifex Desulfurobacterium Sulfur, nitrate reduction Hydrogen oxidation Methanogenesis Carbon fixation Methanocaldococcus Thermovibrio Methanocaldococcus Anemone Mkr 33 Mkr 113 Fortunato et al, Unpub
(Some) Problems with Molecular Methods D/RNA extraction Storage PCR Anytime Incomplete sampling Resistance to cell lysis Enzymatic degradation Inhibitors in template DNA Amplification bias Gene copy number Fidelity of PCR Differential denaturation efficiency Chimeric PCR products Contamination w/ non-target DNA
And the list goes on Optical tweezers Single cell genomics Meta-proteomics Microarrays Flow Cytometry Nano-SIMS FISH In-situ PCR and FISH