Molecular Approaches for Geobiology

This is an introductory lecture on the use of molecular approaches to study microbial assemblages focused on students of the International Geobiology Course in 2014.

Transcript

1. Molecular  Approaches  to   Geobiology   Making  use  of  the  molecular   toolbox   B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma  
2. Nerd   •  Only  takes  notes,  makes   measurements   •  “Brad  and  Blake  at  each   molecular  sampling  site”   Naturalist   •  Swept  up  in  beauty  of  nature   •  “Frank  and  Russell  in  presence  of   stromatolites  they  have  studied   before”   Spaz   •  “Frank  and  Russell  at  new   stromatolite  site”   •  “Bran  and  Blake  at  new   microbial  mat  site”  
3. Environmental   characterisFcs   Ecosystem   fluxes   Diversity   FuncFon   Microbial  Community   field   measurements   B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma  
4. Why  are  the  microbes  there?   •  Who  are  they?   –  Diversity,  ComposiFon,  “Structure”   •  What  are  they  doing?   –  FuncFon   •  How  could/do  they  interact  with  environment?   –  Structure    -­‐-­‐Emergent  property?  
5. Microbiomes   •  PopulaFon  density   •  Community  composiFon   •  Community  structure   •  PhylogeneFc  relatedness   •  Metabolic  capabiliFes   •  Ecological  funcFon   Ecology  (comparisons)   •  SpaFal  distribuFon   •  Temporal  changes   •  Chemical  gradients   •  Macro/microbial  hosts   Why  are  the  microbes  there?  
6. B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma   Diversity   α:  biodiversity  within  area,  community,  ecosystem   •  Species  richness,  #  of  taxa   •  Strongly  influenced  by  sample  size  
7. B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma   Diversity   α:  biodiversity  within  area,  community,  ecosystem   •  Species  richness,  #  of  taxa   •  Strongly  influenced  by  sample  size   β:  diversity  between  ecosystems  or  along  gradients   •  Number  of  taxa  unique  to  each  ecosystem  
8. B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma   Diversity   α:  biodiversity  within  area,  community,  ecosystem   •  Species  richness,  #  of  taxa   •  Strongly  influenced  by  sample  size   β:  diversity  between  ecosystems  or  along  gradients   •  Number  of  taxa  unique  to  each  ecosystem   γ:  total  species  richness  over  a  large  area  or  region   •  Product  of  α  diversity  of  component  ecosystems  and  β   diversity  between  them  
9. Establishing  Boundaries   LHC4 LHC1 LHC3 Little Hot Creek •  What  are  the  boundaries  of  the  system?   – Environmental  parameters   – EnergeFcs   – Physical  structure  
10. Establishing  Boundaries   •  What  are  the  boundaries  of  the  system?   – Environmental  parameters   – EnergeFcs   – Physical  structure  
11. The  Approach   DNA   RNA   Protein   Metabolites  
12. Central  Dogma   DNA   DuplicaFon   RNA  Polymerase   RNA  AU/GC  =  DNA  AT/GC   TranscripFon   mRNA   TranslaFon   Protein   DNA   Polymerase   tRNA-­‐amino  acid   rRNA  (ribosome)   Ribosome  
13. What  makes  a  good  molecular  marker?   •   rRNAs  are  evoluFonarily  conserved   •   PhylogeneFc  inferences  based  on  rRNA   sequence  data   •   Tool  for  taxonomic  and  evoluFonary   study   •  Present in all organisms •  Homologous (common evolutionary origin) •  Sufficiently constrained •  Lack lateral transfer •  Readily obtained and sequenced The  Ribosome  
14. Secondary  Structure:  small  subunit  ribosomal  RNA  
15. Secondary  Structure:  small  subunit  ribosomal  RNA  
16. A G G A C - G A - U G - C G - C A G G A C - G G - C C - G U - A          Primary  sequence  can  change   while  secondary  structure  is  preserved  
17. Universal  PhylogeneAc  Tree     Primary implications: •  Three primary lines of evolutionary descent •  Majority of the tree space occupied by microbes •  Endosymbiotic origins of chloroplasts and mitochondria •  Descendents of a common ancestor share traits Critical assumptions: •  There is a meaningful phylogeny of microorganisms •  rRNA sequences are a reasonable measure of organismal phylogeny
18. Potential effect of lateral gene transfer on organismal phylogeny Phylogenetic Classification and the Universal Tree W. Ford Doolittle 25 June 1999 Science 284: 2124-2128
19. PhylogeneFc  Relatedness   Tree of Life Project (ToL):(http://tolweb.org/tree/) LUCA:  Patrick  Forterre,  circa  1999-­‐2001   (h`p://www-­‐archbac.u-­‐psud.fr/MeeFngs/LesTreilles/LesTreilles_e.html)  
20. The  Approach  
21. The  Approach  
22. The  Approach  
23. DNA/RNA&Extrac-on& Shotgun& Metagenomic& Sequencing& rRNA&Gene& Amplicon& Sequencing& PCR& Barcoding& Quan-fica-on& Pooling& Quan-fica-on& Library& Prepara-on& Library& Prepara-on& Soil& Water& Biofilms& Sample&Collec-on/Preserva-on& The  Approach   •  Where/how  do  you  sample?   •  What  do  you  do  with  it?   •  What  informaFon  is  wanted?   •  What  is  best  approach?  
25. B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma   LimitaFons  to  consider:   •  Bias  in  cell  lysis   •  Amount  of  biomass   •  Co-­‐extracted  inhibitors   The  Approach   1.  Extract  DNA  from  environmental  samples   •  Zymo  XpediFon  Soil/Fecal  DNA  purificaFon  kit   •  Cell  disrupFon/lysis  (bead  beaFng)   •  Removal  of  contaminaFng  organic/inorganic  substances   •  Binding  of  DNA  (ion  exchange)   •  Washing  DNA  (remove  contaminants)   •  EluFon  of  DNA  (low  ionic  strength  soluFon)    
26. 2.  PCR  amplify  genes  of  interest   •  PCR  primers  (gene  specific,  taxa  general/specific)   B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma   The  Approach  
27. Polymerase  Chain  ReacFon  (PCR)   Anatomy  of  PCR:   •   Buffer   •   MgCl2   •   Primers  (forward  and   reverse)   •   deoxynucleoFdes  (dATP,   dCTP,  dGTP,  dTTP)   • Taq  DNA  polymerase   • DNA  template   B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma  
28. 5’   3’   3’   5’   Double  stranded  DNA  -­‐  note  anFparallel  strands   B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma  
29. AddiFon  of  deoxyribonucleoFde   triphosphate  at  3’  end  of  DNA  chain   DNA  polymerase  splits  off  last  two   phosphates  as  pyrophosphate  (PPi )     DNA  polymerase  adds  to  DNA  chain  in  5’  to  3’  direcFon   Nucleic  acid  structure:  h`p://arbl.cvmbs.colostate.edu/hbooks/geneFcs/biotech/basics/nastruct.html   B.  Stevenson,  bradley.stevenson@ou.edu,  University  of  Oklahoma  
30. •   Amplifies  DNAs  by  repeated   rounds  of  DNA  replicaFon  in   vitro   •   DNA  polymerase  (thermally   stable);    “Taq”  DNA   polymerase  (Thermus   aqua4cus)   • Components  of  reac4on:   • Buffer   • dNTPs   • oligonucleo4de  primers   (forward/reverse)   • Taq  DNA  polymerase   • DNA  template  
32. Gel  Electrophoresis   •  electrical  field  in  gel   matrix   •  linear  molecules   separate  by  size   •  agarose:  low  res,  broad   range       •  polyacrylamide:  high  res,   narrow  range   •  DNA  is  negaFvely   charged  -­‐-­‐  migrates   towards  posiFve   electrode   •  VisualizaFon  with   fluorescent  dyes  (e.g.   Ethidium  bromide)   Fig  20-­‐1   Amplified  product   Correct  size   No  other  bands  (gel  purificaFon)   CONTROLS!!!  
33. DNA  sequencing   •  Based  on  separaFon  by  size  of  nested   sets  of  DNA  molecules   •  Each  starts  at  common  5’  end  but   terminates  at  different  3’  posiFon   •  DNA  polymerase  reacFon  (one  primer,   polymerase,  dNTPs,  ddNTPs)   •  IncorporaFon  of  chain-­‐terminaFng   nucleoFdes  stops  fragment  extension  
34. DNA  sequencing   http://www.pcrguru.com/multimedia.html"
35. DNA  Sequencing   •  Fluorescently  labeled  ddNTPs   •  ddA,  ddT,  ddG,  ddC   •  ReacFon  occurs  in  single  tube   •  Fragment  analysis  is  performed  on  a   sequencing  machine,  ‘“sequenator”   •  Fragments  separated  on  polyacrylamide  gel   •  Laser  excites  fluorescent  label,  detector   registers  signal  
36. DNA  sequencing  via  Illumina   •  SFll  based  on  sequencing  through  synthesis   •  Uses  fluorescently-­‐labeled  deoxynucleoFdes  (like  Sanger)   •  Aoer  each  nucleoFde  is  incorporated,  the  synthesis  is  imaged  (i.e.   “analysis  of  color  space”)   •  Fluorescent  moiety  is  enzymaFcally  cleaved  to  allow  for  next   nucleoFde  incorporaFon   Sample  preparaFon   1.  Shear  DNA  to  uniform  size  range  (150-­‐300  bp)   2.  Add  unique  adaptor  oligonucleoFdes   3.  Amplified  using  limited  cycles  of  PCR  with  P5   and  P7  primer  
37. DNA  sequencing  via  Illumina   Cluster  amplificaFon   1.  Add  DNA  to  plate,  hybridizaFon  to  oligonucleoFdes  in  flow  cell    
38. DNA  sequencing  via  Illumina   Cluster  amplificaFon   1.  Add  DNA  to  plate,  hybridizaFon  to  oligonucleoFdes  in  flow  cell     2.  Add  reagents  for  polymerase-­‐based  extension  
39. DNA  sequencing  via  Illumina   Cluster  amplificaFon   1.  Add  DNA  to  plate,  hybridizaFon  to  oligonucleoFdes  in  flow  cell     2.  Add  reagents  for  polymerase-­‐based  extension   3.  Repeated  denaturaFon  and  extension  –millions  of  locaFons  across  flow  cell  surface  
40. DNA  sequencing  via  Illumina   Sequencing   1.  First  cycle  begins  by  adding  fourlabeled  reversable  terminators,  primers,  and  DNA   polymerase   2.  Laser  excites  fluorescent  label  and  each  cluster  is  captured  
41. DNA  sequencing  via  Illumina   Sequencing   1.  First  cycle  begins  by  adding  fourlabeled  reversable  terminators,  primers,  and  DNA   polymerase   2.  Laser  excites  fluorescent  label  and  each  cluster  is  captured   3.  ReacFon  is  repeated…  
42. DNA  sequencing  via  Illumina   Sequencing   1.  First  cycle  begins  by  adding  fourlabeled  reversable  terminators,  primers,  and  DNA   polymerase   2.  Laser  excites  fluorescent  label  and  each  cluster  is  captured   3.  ReacFon  is  repeated…