MetagenomicsMetagenomics: DNA : DNA Sequencing of Sequencing of Environmental SamplesEnvironmental Samples

Susannah Green Susannah Green TringeTringe and Edward M. Rubinand Edward M. RubinDepartment of Energy Joint Genome InstituteDepartment of Energy Joint Genome Institute

What is What is MetagenomicsMetagenomics??

MetagenomicsMetagenomics is the study of genomes from whole is the study of genomes from whole communities rather than individual speciescommunities rather than individual species–– recent advances and decreases in cost have allowed recent advances and decreases in cost have allowed

biologists to study genomes of organisms that cannot biologists to study genomes of organisms that cannot survive on their own, such as survive on their own, such as symbiontssymbionts and pathogens and pathogens

This review focuses on methodological advances This review focuses on methodological advances that have allowed the sequencing of natural that have allowed the sequencing of natural populations, examples of these techniques, and populations, examples of these techniques, and possible future directions for research.possible future directions for research.

Methodological AdvancesMethodological Advances

Natural samples contain DNA in Natural samples contain DNA in several formsseveral forms–– free DNA, virus particles, cells, etc.free DNA, virus particles, cells, etc.

These samples can be suspended in These samples can be suspended in water, bound to the soil or other solid water, bound to the soil or other solid particles, or contained within an particles, or contained within an aggregate of microorganisms (aggregate of microorganisms (biofilmbiofilm))

Methodological AdvancesMethodological Advances

Water suspended (aquatic) samples Water suspended (aquatic) samples are easier to concentrate than solidare easier to concentrate than solid--bound particlesbound particles–– may be filtered before concentrationmay be filtered before concentration

choose filtration size by type of particleschoose filtration size by type of particleschoose filtration based on either eukaryotic, choose filtration based on either eukaryotic, prokaryotic, or viral particlesprokaryotic, or viral particles

–– concentrated by normal or tangential flow concentrated by normal or tangential flow filtrationfiltration

Methodological AdvancesMethodological Advances

SoilSoil--suspended particles often contain suspended particles often contain enzyme inhibiting substancesenzyme inhibiting substances–– DNA isolation is either direct (cells DNA isolation is either direct (cells lysedlysed within within

sample material) or indirect (separated before sample material) or indirect (separated before lysinglysing))

lysinglysing technique can change what DNA is extractedtechnique can change what DNA is extractedi.e. grami.e. gram--positive bacteria are hard to positive bacteria are hard to lyselyse

–– contaminants can be removed with contaminants can be removed with agaroseagarose gel gel electrophoresis or chromatography methodselectrophoresis or chromatography methods

Once DNA is extracted, it can be clonedOnce DNA is extracted, it can be cloned

Example: 16S Example: 16S rRNArRNA

99% of microbes are not easily 99% of microbes are not easily culturedculturedAll these microbes have 16S/18S SSU All these microbes have 16S/18S SSU rRNArRNA–– can be used to determine phylogenycan be used to determine phylogeny

Used largeUsed large--insert clone sequencing insert clone sequencing Discovery of Discovery of proteorhodopsinproteorhodopsin in in oceanic bacteriaoceanic bacteria–– previously thought to be dependent previously thought to be dependent

on organic matter instead of lighton organic matter instead of light–– first time a first time a rhodopsinrhodopsin--like protein like protein

had been discovered in the had been discovered in the microorganism domainmicroorganism domain

Evidence for horizontal gene transfer Evidence for horizontal gene transfer (exchange of genetic material (exchange of genetic material between two genomes without a between two genomes without a parental relationship)parental relationship)

Example:HostExample:Host--Associated Associated BacteriaBacteria

TreponemaTreponema pallidumpallidumcauses syphiliscauses syphilis

–– obtained from the obtained from the testes of rabbits testes of rabbits through through lysislysis and and centrifugationcentrifugation

–– analysis aided with analysis aided with virulence aspects virulence aspects of bacteriaof bacteria

Whipple disease is Whipple disease is caused by caused by TropherymaTropheryma whippleiwhipplei

–– analysis revealed analysis revealed reasons for reasons for inability to culture inability to culture and was corrected and was corrected with amino acid with amino acid and tissue culture and tissue culture regimeregime

BacteriomesBacteriomes(specialized organs) (specialized organs) from from BuchneraBuchneraamphidicolaamphidicola were were isolated using isolated using dissection, dissection, differential differential lysislysis, and , and pulsepulse--field field electrophoresiselectrophoresis

Example: Example: PaleogenomicsPaleogenomics

By using ancient DNA By using ancient DNA (as much as 50,000 (as much as 50,000 years), scientists can years), scientists can determine phylogeny of determine phylogeny of extinct species through extinct species through genetics rather than genetics rather than solely on morphologysolely on morphology

–– with with metagenomicsmetagenomics, it , it doesndoesn’’t matter that t matter that the DNA is the DNA is contaminated by contaminated by microbesmicrobes

40,000 year old cave 40,000 year old cave bear was studiedbear was studied

–– only 27 kb of genome only 27 kb of genome was sequenced, but was sequenced, but enough to compare it enough to compare it to black and brown to black and brown bearsbears

Possibility of looking at Possibility of looking at NeaderthalsNeaderthals

Example: Shotgun Example: Shotgun SequencingSequencing

AcidAcid--mine mine biofilmbiofilm–– small number of species (small number of species (chemilithotrophschemilithotrophs) can survive in highly acidic and metal filled ) can survive in highly acidic and metal filled

environmentenvironment–– 76.2 million 76.2 million bpbp from this communityfrom this community–– 2 near2 near--complete genomes and 3 partial genomescomplete genomes and 3 partial genomes–– LeptospirillumLeptospirillum is the only species in community is the only species in community

that can fix nitrogenthat can fix nitrogenpossibilities for acid mine drainage controls possibilities for acid mine drainage controls

Sargasso SeaSargasso Sea--low nutrient levellow nutrient level–– 7 libraries; 1.6 7 libraries; 1.6 GbGb DNA from this studyDNA from this study–– only 3% of this was accounted for only 3% of this was accounted for –– 1.2 million genes found similar genes in database1.2 million genes found similar genes in database–– less than 1/3 could be assigned to a cellular roleless than 1/3 could be assigned to a cellular role–– most genes couldnmost genes couldn’’t be assigned to t be assigned to phylogeneticphylogenetic

groupgroup–– encode for phosphorus uptake, encode for phosphorus uptake, proteorhodopsinproteorhodopsin, ,

and many othersand many othersNutrient rich environmentsNutrient rich environments

–– agricultural soil and whale fallsagricultural soil and whale falls–– couldncouldn’’t complete genomes because of high t complete genomes because of high

number of organisms, but identified gene number of organisms, but identified gene families of importancefamilies of importance

environmental gene tag (EGT)environmental gene tag (EGT)

Future DirectionsFuture Directions

““Second human genome projectSecond human genome project”” to study the microorganisms in our bodiesto study the microorganisms in our bodiesCurrent methods of DNA isolation, library construction, sequenciCurrent methods of DNA isolation, library construction, sequencing, and ng, and analysis will have to be reworked from a analysis will have to be reworked from a metagenomicsmetagenomics standpointstandpoint

–– techniques have been used to obtain samples from all representedtechniques have been used to obtain samples from all represented organisms in the organisms in the communitycommunity

great for overviews of community and determining dominant factorgreat for overviews of community and determining dominant factorsstrouble if trying to obtain complete genome sequencetrouble if trying to obtain complete genome sequence

Separation techniques have been developedSeparation techniques have been developed–– filtration, fractionation based on GC content, differential centfiltration, fractionation based on GC content, differential centrifugation, density rifugation, density

gradients, differential gradients, differential lysislysis, pulsed, pulsed--field electrophoresis, and selective use of field electrophoresis, and selective use of restriction enzymesrestriction enzymes

Interest in species that perform specific metabolismInterest in species that perform specific metabolism–– use of stable isotope probing to isolate organismsuse of stable isotope probing to isolate organisms–– flow flow cytometrycytometry (measures fluorescence) isolates organisms by viability, membra(measures fluorescence) isolates organisms by viability, membrane ne

properties, surface protein expression, or SSU properties, surface protein expression, or SSU rRNArRNA sequencesequence–– affinity purificationaffinity purification--uses binding of protein, possibly cell wall proteins, on solid uses binding of protein, possibly cell wall proteins, on solid

surface to separatesurface to separateIsothermal strand displacement is DNA amplification that doesnIsothermal strand displacement is DNA amplification that doesn’’t require a t require a large amount of starting materiallarge amount of starting material

–– could be used for whole genome studiescould be used for whole genome studiesLibrary construction with Library construction with pyrosequencingpyrosequencing (detect pyrophosphate) to sequence (detect pyrophosphate) to sequence rather than cloning to eliminate rather than cloning to eliminate EE.. colicoli biasbias

Future DirectionsFuture Directions

Genome assembly is importantGenome assembly is important–– has been shown that cross species assemblies are has been shown that cross species assemblies are

uncommon except for highly conserved and uncommon except for highly conserved and rRNArRNAsequencessequences

–– effects of heterogeneity on assembly procedures should be effects of heterogeneity on assembly procedures should be studiesstudies

could there be correlation with growth rate, competition, could there be correlation with growth rate, competition, community stability, etc.community stability, etc.

Assigning these sequences to phylogenic trees Assigning these sequences to phylogenic trees should be done with cautionshould be done with caution–– use related species to help with processuse related species to help with process

Biggest advances will occur with more fully Biggest advances will occur with more fully sequenced genomes sequenced genomes

Conclusions & CommentsConclusions & Comments

MetagenomicsMetagenomics offers information about offers information about populations rather than individuals, which is populations rather than individuals, which is more representative of the natural worldmore representative of the natural worldWhy is there a limit on age for Why is there a limit on age for paleogenomicspaleogenomics??How many genes are highly conserved and How many genes are highly conserved and can these ever truly be used in can these ever truly be used in metagenomicmetagenomic studies?studies?