More than 70 projects wereselected for the 2010 Com-munity Sequencing Program(CSP) portfolio. They involveorganisms from regions as farnorth as the Arctic and southto New Zealand. They includeproposals to study microbialcontaminants in alcohol thatcould impact biofuel production,microbial communities in theguts of insects from an areageography scholar JaredDiamond once described as“the nearest approach to lifeon another planet” and anovel bacterial isolate that

could be used to remove heavymetal contaminants from fresh-water streams.

“The information we generatefrom these projects promisesto improve the clean, renew-able energy pathways beingdeveloped now as well as lendresearchers more insight intothe global carbon cycle, optionsfor bioremediation, and biogeo-chemical processes,” said DOEJGI Director Eddy Rubin. “Intranslating DNA sequence datainto biology, we generate valu-able science that improves ourunderstanding of the complex

processes that support life onthe planet, or imperil it.”

Following along the lines ofprevious CSP-approved meta-genomic projects involving the

cow, termite and Amazonianstinkbird, Mike Taylor fromNew Zealand’s University ofAuckland proposed sequencingmicrobial communities inside

BY MASSIE SANTOS BALLON

To get an idea of how manymicrobes there are on Earth,imagine taking just the small-est type of microbe, the virus-es that infect other microbes,and lining them all up in therow. How far do you think thatline would reach? From SanFrancisco to Paris? To themoon? To the sun? Theanswer: A thousand times thedistance across the Milky Way.

Microbes are not onlyexceedingly numerous, butalso extremely diverse, encom-passing most of Earth’s totalbiodiversity. This is one reason

why two-thirds of the nearly5,000 genome projects report-ed in the Genomes OnLineDatabase involve microbes.Another factor is the myriadpotential applications of micro-bial research for targeted drugdevelopment, bioenergy, biore-mediation, agriculture, andother human endeavors. Butfar more could be done withmicrobial genomics, accordingto DOE JGI Genome Biologyhead Nikos Kyrpides, ifresearchers would embracethe world of possibilities thatextend beyond such human-centric studies.

In an article published inthe July issue of the journalNature Biotechnology, Kyrpidesreflects on the role of microbialstudies in the genomics revo-lution of the past decade, andconsiders the factors that havehindered the advancement ofthe field. Although nearly1,000 microbial genomes havebeen sequenced over the past15 years, he noted that thedata obtained has been com-promised by the lack of a com-mon language and standard-ized procedures. Such stan-dards, required for theexchange and integration of

genomic data, still remain tobe defined and codified. Dueto biases in the selection ofgenomes for study, vast realmsof biodiversity remain unex-plored. Data release policiesvery early on led to what heterms “a violation of a long-standing precept in all scientif-ic fields” when reviewers ofsubmitted publicationscouldn’t access the underlyinggenomic data.

Kyrpides offers numeroussuggestions to meet theseand other challenges that facegenomics research in thedecade ahead. For example,

Bugs, Big & Small, fromInside & Out, Join Sources ofGreen Genes to Fill CSP2010 Pipeline

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On Sequencing, Finishing andAnalysis in Santa Fe . . . . . . 2

Supporting Life in Great SaltLake . . . . . . . . . . . . . . . . . . . 3

Brown-rot for Biofuels . . . . . 4

Sequencing with a Single Cell . . . . . . . . . . . . . . 5

Notes from the 4th Annual JGIUser Meeting . . . . . . . . . . 6-7

Improving Crops andFeedstocks . . . . . . . . . . . . . 8

A Shortcut called ChIP-seq 9

Algae as a climate changesensor . . . . . . . . . . . . . . . . 10

also in this issue

Moving Microbial Genomics Forward

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BY MASSIE SANTOS BALLON

One thing became clear fairly quickly atthe 4th Sequencing, Finishing, Analysis inthe Future Meeting held in Santa Fe, NMfrom May 27-29, 2009: the name gamegenome researchers play needs new rules.

Speaking immediately after the openingkeynote, Patrick Chain, head of MicrobialInteractions program at DOE JGI, noted thatthere have traditionally been only two cate-gories for sequenced genomes — “draft,”where the genetic data of an organism hasbeen collected but not completely organized,or “finished,” in which the genetic code hasbeen correctly put together and can be readlike a published book from beginning to end.He then proposed adding four other labelsso that researchers can determine thequality of the genomes they’re accessing.

Unfortunately, Chain said, pitching theidea on behalf of the International GenomeSequencing Standards Consortium, theadvent of new sequencing technologieshas increased the number of genomesbeing sequenced every year.

“Data generation is becoming cheaper,easier and faster,” he said, ”and this is onlygetting worse.” Based on the current output,Chain predicted that by 2012, there might be12,000 incomplete genome sequences beingmade available to researchers who mightnot recognize that the datasets may lackthe information they’re interested in using.

Chain said another part of the problemis that as new sequencing technologiesbecome the machines of choice, they needto be evaluated so that researchers under-stand that the information that comes outof one company’s instrument may not bethe same as the information provided byother equipment. The tags applied to thedata may also vary based on the sequenc-ing technology used.

All of these changes, Chain said, haveled to the Consortium’s proposal of fourgenome quality categories, as well as somesuggested changes to the definitions of thetraditional terms, none of which are peggedto a particular sequencing technology. Astandard draft, for example, might be

something produced in a lab other than alarge sequencing center such as DOE JGI,which typically generates what he called a“high quality draft,” which has little or nomanual review.

In an “improved high quality draft,” thedata has been reviewed both by people andmachines to some extent so most of thegenetic data is assembled correctly, butsome errors may still be present. An“annotation grade” sequence would presentall the information in various gene regionsas accurately as possible, and the term“non-contiguous finished” would apply togenomes that are practically finished exceptfor “recalcitrant regions” that are provingproblematic. Such quirks, Chain said,would be included in the comments whenthe genome sequence is made available.

It’s worth wondering after all this what agenome sequence has to do to be labeled“finished.” To achieve this pinnacle, Chainsaid, the sequence would have no morethan one error in every 100,000 base pairs.

Chain noted that one of the hardestparts of coming up with categories ofsequence status had been getting every-one in the consortium to agree on theterms and definitions. “I can barely getagreement in my own group on some ofthese issues so this was really a fantasticachievement,” he said. He concluded bynoting that the Consortium plans to submittheir proposal for publication at a laterdate, and intends to publicize the stan-dards on the websites of the various insti-tutions affiliated with the group.

A number of other speakers referencedthe need for a standardized terminologythat could describe a genome sequenceand its various components in their talksover the three days of the conference.Among them was Deanna Church from theNational Center of Biotechnology Information,who referred to the need for standardterms during her talk when she mentioned

Meeting organizers (left to right): Michael Fitzgerald, Broad Institute; Patrick Chain, DOEJGI; Bob Fulton, Washington University in St. Louis; Donna Muzny, Baylor College ofMedicine; Johar Ali, Ontario Institute for Cancer Research; Chris Detter, DOE JGI; AllaLapidus, DOE JGI

Seeking Sequencing Standards in Santa Fe

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BY MADOLYN BOWMAN ROGERS

In one of the 2009 CommunitySequencing Projects, the DOE JGI arepartnering with a consortium of researchersto sequence the genomes of microbialcommunities in Utah’s Great Salt Lake.Researchers believe the lake’s harsh envi-ronment harbors a wealth of undiscoveredbiodiversity that may provide new solu-tions for carbon sequestration and biore-mediation, and might even give clues tothe nature of life on other planets.

“We believe we’re going to find newenzymes and new metabolic routes thathave never been seen before,” said theproject’s lead investigator, Bart Weimer, amicrobiologist at University of California,Davis. The collaboration also includesresearchers at the Utah State University,the U.S. Geological Survey, and the UtahDivision of Water Quality.

The Great Salt Lake is the third saltiestbody of water on earth, and the saltiest tosupport life. The salt content varies widelyacross the lake, but on average the lakeis four times saltier than the ocean, andin the North Arm up to 20 times saltier.The lake also contains petroleum seeps,

mercury and other heavy metals, and highvolatile sulfur concentrations that give thelake a distinctive rotten-eggs stink.Despite these compounds, the lake sup-ports five million migrating birds each yearand is home to brine shrimp and severalkinds of algae including diatoms, as wellas an untapped diversity of microbial life.

Because the microbes can survive insuch an extreme environment, Weimerbelieves they may contain unique proteinsthat enable them to chemically fix sulfurand carbon and to detoxify pollutants.Their biochemistry may suggest new meth-ods for sequestering carbon and reducingacid rain, Weimer said, as well as novelpathways for bioremediation of heavy met-als, aromatic hydrocarbons, chlorinatedcompounds and methylmercury (Weimer etal., 2009). Microbial life in the Great SaltLake might also provide a model for lifeon planets such as Mars, where sulfateand salt concentrations are also very high.

Since less than one percent of themicrobes in the lake can be cultured,most of the microorganisms have neverbeen studied before. The DOE JGI willsequence DNA from four sites in the lake

to provide a baseline picture of microbialdiversity. Researchers will combine theDNA data with measurements of microbialmetabolites and environmental conditionssuch as salinity and oxygen to paint acomplete picture of the lake’s ecology.

“We’ve done a lot of diversity assess-ment of the lake in the last two years,and we’re not even approaching the limitof microbial diversity,” Weimer said. “Theservices that the DOE JGI will provide aregoing to eclipse anything that we could doin the next five to ten years.”

FOR ADDITIONAL INFORMATION SEE:

Weimer, Bart C., Giovanni Rompato,Jacob Parnell, Reed Gann, BalasubramanianGanesan, Cristian Navas, Martin Gonzalez,Mario Clavel, Steven Albee-Scott. Microbialbiodiversity of Great Salt Lake, Utah.2009. In Saline lakes around the world:unique systems with unique values.pp.15-22. Eds. Oren, A., D. L. Naftz, andW. A. Wurtsbaugh. S. J. and Jessie E.Quinney Natural Resources ResearchLibrary, Utah State University Press,Logan. (http://www.cnr.usu.edu/quin-ney/htm/publications/nrei)

Salt Lake’s BrinyWaters May HarborBiological Treasures

Photo by Charles Uibel, GreatSaltLakePhotos.com

Lignin is to plants what a skeleton isfor humans: an internal scaffold that keepsthe organism upright and the softer insidesprotected. As a result, one of the prob-lems with making biofuels from plants isgetting past the lignin to break down thecellulosic biomass into sugars that canbe fermented and distilled into liquid bio-fuel. Because the lignin prevents easyaccess to the cellulosic material, breakingdown plant biomass has traditionallyinvolved harsh chemicals and high heat,processes which add energy to the wrongside of the biofuel production equation.

The Department of Energy (DOE) andits Bioenergy Research Centers areapproaching the problem from two differ-ent angles: developing plants bred specifi-cally to become biofuel feedstocks; andidentifying enzymes that can maximize thebiomass being converted into fuel.

With the latter idea in mind, an interna-tional team led by researchers from theDOE JGI examined the complete genetic

code of brown-rot fungi to find out howthey can harness the fungi’s ability tobreak down wood to make biofuel produc-tion more cost-effective.

“The microbial world represents a littleexplored yet bountiful resource for enzymesthat can play a central role in the decon-struction of plant biomass—an early stepin biofuel production,” said Eddy Rubin,Director of the DOE JGI, where the brown-rot fungi’s genome was sequenced. “Thebrown-rot Postia placenta’s genome offersus a detailed inventory of the biomass-degrading enzymes that this and otherfungi possess.”

Brown-rot fungi affect softwood treessuch as redwoods, pines, cedars andDouglas firs, removing practically all of thecellulose in the plant without removing thelignin as well. Interestingly, some researchershave noted that most brown-rot fungi can’twork on cellulose if the lignin is missing.Approximately 10 percent of the decay notedannually in American timber is attributed

to brown-rot fungi, which makes the woodlook like a pile of cracked bricks.Researchers hope to identify the enzymesinvolved in attacking the wood and applythem toward other plant material.

“Nature offers some guidance here,”said Dan Cullen, U.S. Department ofAgriculture Forest Service, Forest ProductsLaboratory (FPL) scientist and one of thesenior authors on the PNAS paper.“Postia has, over its evolution, shed theconventional enzymatic machinery forattacking plant material. Instead, the evi-dence suggests that it utilizes an arsenalof small oxidizing agents that blast throughplant cell walls to depolymerize the cellu-lose. This biological process opens a doorto more effective, less-energy intensiveand more environmentally-sound strategiesfor more lignocellulose deconstruction.”

Randy Berka, another of the study’ssenior authors and Director of IntegrativeBiology at Davis, Calif.-based Novozymes,Inc., noted that in sequencing the genomeof brown-rot fungi, researchers have beenable to compare the genetic blueprints ofvarious types of wood-decaying fungi forthe first time.

Brown-rot fungi is a type of basid-iomycete fungi like white-rot fungi, whichhad been previously studied by the DOEJGI. White-rot fungi degrades both thelignin and the cellulose however, whilebrown-rot fungi only works on the latter.

“Such comparisons will increase ourunderstanding of the diverse mechanismsand chemistries involved in lignocellulosedegradation,” Berka said. “This type of infor-mation may empower industrial biotechnol-ogists to devise new strategies to enhanceefficiencies and reduce costs associatedwith biomass conversion for renewablefuels and chemical intermediates.”

The report by appeared in the February4 online edition of the Proceedings of theNational Academy of Sciences (PNAS) andinvolved over 50 scientists from Austria,Canada, Chile, the Czech Republic, France,Germany, Spain and the United States.

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Studying microbes in a lab setting usu-ally involves growing large quantities ofthem for genetic studies. Researchers atthe DOE JGI demonstrated they couldassemble a high quality draft of twomarine microorganisms using just a singlecell from each.

“As long as you can isolate a singlecell, pick it from the environment, lyse it(blow it open), you can generate millionsof copies of that genome and gain accessto the information inside that organism,”said DOE JGI researcher Tanja Woyke (pic-tured, top right).

Woyke and her colleagues at the DOEJGI sequenced genomes of two uncul-tured flavobacteria, marine microorgan-isms collected in Maine’s BoothbayHarbor for Bigelow Laboratory collabora-tors Ramunas Stepanauskas and MichaelSieracki, who are particularly interested ingenes encoding proteorhodopsins, whichallow some cells to use energy from thesun without applying photosynthesis.

After the Bigelow scientists had pickedout individual bacterial cells from theirbacterial samples, Woyke and her team

went to work. The single cells were thenblown open and a process called multipledisplacement amplification was applied tomake millions of copies of the bacterialgenomes for sequencing.

The resulting flavobacterial genomesequences are approximately 80 to 90percent complete. Woyke credited DOEJGI’s Cliff Han and his team at LosAlamos National Laboratory (LANL), whichworked on closing gaps in the assembly.

“Even without completed genomeassemblies, single cell sequencing offersradically new opportunities for the basicresearch and biotechnology applications ofthe microbial ‘uncultured majority’,” saidDOE JGI collaborator Stepanauskas.

More importantly, the single cellsequencing approach means researchersdon’t have to culture organisms in orderto sequence them. This could prove usefulfor studying organisms that can’t exist ina lab environment.

“We estimate that roughly 99.9 percentof the microbes that exist on this planetcurrently elude standard culturing methods,denying us access to their genetic material,so we have to explore other methods tocharacterize them,” noted DOE JGI DirectorEddy Rubin. As a national user facility, hesaid, the DOE JGI is dedicated to helping

researchers access genomes of interestthat are relevant to the DOE’s mission.

“The power of single cell genomics isthat it offers us the ability to sort out onecell from a complex environmental sam-ple, liberate the DNA from that cell, andenzymatically produce millions of copiesof that genome so that we have enoughDNA to sequence it and characterize itsmetabolic potential,” he said.

The technique doesn’t just work onmarine microorganisms either; Woyke saidthey are helping other DOE JGI collabora-tors who study organisms from variousenvironments but can’t provide enoughsample to apply other test methods.

Woyke and her colleagues aren’t thefirst to work on the single cell genomesequencing technique, but they’ve beenable to provide a higher quality resultcompared to previous efforts. Still, themethod needs more work. “One of thekey issues that still needs refining is thelysis step, since many microbes will notlyse with alkaline solutions, the mostcommon agent for the job. But we areactively working on that,” she said.

The study was published in the April 23edition of the journal PLoS One. TanjaWoyke can be heard discussing single cellgenome sequencing as it applies to thisparticular project and other points of inter-est at http://www.jgi.doe.gov/News/pod-cast090421_woyke.mp3.

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Coastal water sample from Boothbay Harbor, Maine collected by Bigelow Laboratoryteam. Photo courtesy of Ramunas Stepanauskas (Bigelow Laboratory).

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A record 500 people attended the DOEJGI Annual Genomics of Energy andEnvironment User Meeting held March 25-27, 2009 at the Marriott Hotel in WalnutCreek, Calif.

This year the keynote speakers wereEnergy Bioscience Institute director ChrisSomerville, Harvard University’s GeorgeChurch and founder of the eponymousgenomic research institute J. CraigVenter. All three speakers focused on anumber of scientific and technologicaltechniques to move genomic sequencingforward in ways relevant to the DOE JGI’smission of clean energy generation andbioremediation.

Somerville’s talk(http://www.scivee.tv/node/10674)focused on thedevelopment of cel-lulosic biofuels, whichis being heavily pro-moted by the Obama

administration and last year’s keynotespeaker-turned-energy secretary Steven Chu.He spoke about the challenges involved in

harnessing nature, particularly the issue ofcarbon emissions from indirect land use,which has dogged bioenergy researcherssince the concern was raised in a Sciencepaper (see sidebar and Searchinger et al,2008; 319: 1238-1240.) published lastyear. And he painted a sobering picture ofthe timelines involved in modifying plantswhose genomes were being sequenced tobecome better feedstocks, which he con-siders a long-term solution, as well aswhen producing a billion gallons of cellu-losic ethanol annually would likely happen.

During the talk, Somerville remindedpeople to think about the energy problem ona global scale. He also noted that thinkingoutside the box can lead to surprising col-laborations and solutions being proposed.

“We learned it can be very productiveto educate people who don’t know about atopic about what the problems are,” he said.

Church’s keynote(http://www.scivee.tv/node/10578) thefollowing nightfocused on the tech-nologies needed tomove genomicsresearch forward. He

began by noting that the technologyshould be accessible enough that usersfeel comfortable not just using the equip-ment but modifying it to suit their needs.

“Think of the platform that readssequences as an automated microscope.That’s really what it is.” Adding amicromirror display, he said, would givethe machine added functionality, allowingresearchers to hold or release cells orsequences of interest.

The Harvard researcher also describeda program he called Multiplex AutomatedGenome Engineering or MAGE that wouldallow researchers to study and design

sequences “without any idea of where youare or where you’re going at all.” As anexample of how the system could be used,he talked about an agricultural study inwhich lycopene was added to a strain oftomato that lacked the antioxidant.

Venter’s presenta-tion (http://www.scivee.tv/node/10653) on the lastday of the UserMeeting combinedSomerville’s focus ongenome sequencing

to design and develop better bioenergysources with Church’s emphasis on thetechnological upgrades needed to movethe science forward. He spoke about whathe termed “digitizing life,” moving toward

reading the genetic code on the computerand writing it. He discussed the chal-lenges of building a bacterial genomefrom the ground up, which led to thedevelopment of computer software thatallows his researchers to act as digital

JGI Annual User Meeting #4—Briefly In Review

My favorite among these

[energy] crops is Miscanthus. In an

average car you could drive around

the world twice from fuel produced

by one acre of biomass.

There are trillions of dollars

invested in vehicle infrastructure

and to think we’ll switch overnight

to ethanol and hydrogen isn’t nec-

essarily in the cards.

—Chris Somerville

—George Church

If you’re looking for new

mammalian genes, stop. Basically

they’ve all been found.

—J. Craig Venter

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DNA designers to create thespecies needed to meet the ever-increasing demand for energy andthe need to protect the environment.

“We have discovered 20 milliongenes and we’re using them asdesign components of the future,”

Venter said. When asked at theend about making the software orthe species being designed avail-able, he quipped, “We are notready to release our software orour bugs because the softwarehas bugs.”

In a February 2008 issue of Science, TimSearchinger and his colleagues at PrincetonUniversity argued that most studies advocat-ing biofuel use as a way to reduce green-house gas emissions did not factor in theemissions resulting from the conversion ofland to grow the additional energy crops.

The researchers argued that carbon sinkssuch as forests and grasslands would becomecarbon sources when they were plowed up foruse as feedstock farmland. “At the broadestlevel, the key insight of our paper is that whencropland-based feedstocks are used for biofuels,the source of any potential greenhouse gasbenefit is the use of the land to take carbonout of the atmosphere,” Searchinger reiteratedin a letter published by Science six monthsafter the study was released.

“Leaving out emissions from land usechange for such biofuels assumes that land isa carbon-free asset. We would argue that any

calculation that ignores these emissions, howeverchallenging it is to predict them with certainty, is tooincomplete to provide a basis for policy decisions.”

One of the examples Searchinger and his teamused involved corn ethanol. Biofuel advocates had pre-viously calculated that the energy crop would reduceemissions by 62 percent. The Princeton researcherssaid that when land use emissions were factored in,corn ethanol would actually release double the emis-sions that were to have been sequestered.

The study drew criticisms from biofuels advocatesand researchers such as Chris Somerville, who notedthat the study was “based on naïve assumptions.”

The Princeton study, they said, only targeted landuse emissions from biofuel production, instead of alsoconsidering the emissions from oil production. Anotherpoint they raised was that the study assumed a worst-case scenario in which all the land being converted forthe added crops was pristine land. “According to indi-rect land use,” Somerville commented during hiskeynote speech, “by not growing corn on unfarmed land,we’re deforesting the Amazon.” Other factors, the crit-ics said, could make land available for biofuels withoutdestroying forests.

Searchinger and his colleagues have refuted thecounter-arguments, but the debate continues to this day.

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“…here are my top lessons I got out of the meeting…”•NextGen sequencing con-tinues to open up newwindows into biology.

•Ecological and populationgenomics are truly thenext big thing.

•Related to the abovepoint, one of the next rev-olutions is going to be inhigh throughput phenotyp-ing --- after all, we cannotsolve the genotype-phenotype problem when we only know the geno-type.

•Model / reference organisms are still in, but every single organism onthe planet is now in play.

•NextGen sequencing has completely outrun the ability of even goodbioinformatics people to keep up with the data and to use it well.

•Related to the above point, the NextNextGen (e.g., PacificBiosciences) seems to be barreling along and almost ready for primetime. What are we going to do in terms of informatics then?

•Following up on the above point- we desperately need a MASSIVEeffort in the development of tools for "normal" biologists to make bet-ter use of massive sequence databases.

•I am happy to report that just about everyone seems to be trying touse an evolutionary perspective as part of their work - especially inthe selection of organisms for sequencing

•I am sorry to report that many of the evolutionary "perspectives" are abit off kilter.

•If you study a plant or an animal and are not studying the microbesthat live with them, you are missing something.

•If you study ANY organism and are ignoring epigenetics you are behindthe curve

•Reading DNA is being used in every which way imaginable. •Sequencing is definitely not over - it is just getting started.•Next up – writing DNA.

Lessons Learned at the JGI Users Meeting (excerpts from Jonathan Eisen: http://phylogenomics.blogspot.com/2009/03/lessons-learned-at-jgi-users-meeting.html)

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For some 500 million people aroundthe world, sorghum is a staple grain.Approximately 60 million tons of sorghum,which is more drought-tolerant than cornand other cereal crops such as wheat andoats, is produced annually.

Sorghum also requires a third lesswater to produce the same amount ofethanol per bushel compared to corn.Because of its potential as a whole plantfiber based biofuel feedstock, the DOEJGI and several partner institutionsworked on the genome sequence ofsorghum. The grass’ complete genomewas published in the January 29 editionof the journal Nature.

“This is an important step on the roadto the development of cost-effective biofu-els made from nonfood plant fiber,” saidAnna C. Palmisano, DOE Associate Directorof Science for Biological and EnvironmentalResearch.

“Sorghum is an excellent candidate forbiofuels production, with its ability to with-stand drought and prosper on more mar-ginal land,” she said. “The fully sequencedgenome will be an indispensable tool forresearchers seeking to develop plant vari-ants that maximize these benefits.”

Researchers plan to use the geneticdata not only to develop better food andfeedstocks with improved drought toleranceand increased grain yield, but to producegrass strains suited specifically for biofuelproduction. Sorghum’s position in the grassfamily ⎯ it is closely related to maize andsugarcane, and more distantly to wheat andrice ⎯ is expected to give researchershelpful insights into other plant genomesthat could be used as biofuels.

“Sorghum will serve as a templategenome to which the code of the other

important biofuel feedstock grassgenomes⎯switchgrass, Miscanthus, andsugarcane⎯will be compared,” addedAndrew Paterson, the publication’s firstauthor and Director of the Plant GenomeMapping Laboratory, University of Georgia.

Having the complete sorghum genomealready benefits a number of currentresearch projects involving the grain. Evenbefore they knew sorghum’s completegenome sequence, for example, the U.S.Department of Agriculture had been work-ing on developing disease-resistant, low-lignin sorghum strains. USDA researchersbased their studies on early sorghumgenetics research that comparedsorghum’s genes to those of rice.

With less lignin in the plant, the sorghumwould be easier to digest, making it a bet-ter livestock feed. It would also requireless energy to be converted into ethanolbiofuel. Similar research projects in otherinstitutions would find the sorghumgenome’s availability equally useful.

The sorghum genome is only the sec-ond grass genome to be completelysequenced after rice and has turned outto be nearly 75 percent larger. Part of thereason for the difference in size, notedJeremy Schmutz from the DOE JGI partnerHudsonAlpha Institute for Biotechnology,is that plant genomes tend to have largesections of repetitive regions and sorghumis no exception. But the comparisonbetween sorghum and rice genomes sug-gests that there may be a common grassgenome structure as well.

“We found that over 10,000 proposedrice genes are actually just fragments,” saidDOE JGI’s Dan Rokhsar. “We are confidentnow that rice’s gene count is similar tosorghum’s at 30,000, typical of grasses.”

JGI Publishes SorghumDrought-Tolerant Biofuels Feedstock

the PRIMER / 9Summer 2009 Vol. 6, Issue 1

To identify potentially significantsequences in an organism’s genetic code,such as those that control the genesresponsible for an organism’s develop-ment or key the development of betterbiofuel feedstocks, genome researcherstypically compare the target genome withthe genomes of other, similar species andlook for sequences conserved in evolution.

DOE JGI researchers teamed with col-leagues at Lawrence Berkeley NationalLaboratory and the University ofCalifornia, San Diego to develop a toolthat provides genome researchers with ashortcut for searching out locations onthe genome where particular gene func-tions take place. The method is known asChIP-seq and it can pinpoint the geneticswitches by looking at how DNA and pro-teins interact.

As DOE JGI postdoctoral fellowMatthew Blow put it, ChIP-seqallows scientists to directlyidentify a genome-wide setof the genetic switchesthroughout the wholegenome rather than individ-ually tracking down andtesting every gene forits possible functions.

Len Pennacchio,head of the DOE JGI’sGenomic Technologies depart-ment and senior author of the study,added that ChIP-seq also providesresearchers with an opportunity to learnmore about noncoding sequences referredto as genomic “dark matter.”

The research was described in theFebruary 12 edition of the journal Nature.

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A Shortcut for Genome Sleuths

The boxing gloves-like blue stain in this-mouse embryo indicates a specific genefunction identified by researchers using atool refined by (left-to-right) senior Naturepaper author and JGI Genomic TechnologiesProgram head Len Pennacchio and co-firstauthors Matt Blow and Axel Visel.

that the same label might be applied to dif-ferent things on different databases. AndOwen White from University of Marylandsuggested moving toward a common sys-tem that could pull the best information onbacterial genome sequences from a thevarious databases rather than relying onjust a few during his keynote speech.

“Standards are going to be the key tounderstanding some of the differencebetween the things that are done well andthe things that are sort of dubious,” saidMiriam Land, a DOE JGI researcher at OakRidge National Laboratory who annotatesmicrobial genomes sequenced in DOE JGIheadquarters in Walnut Creek, Calif.

Land was the final speaker at the confer-ence and she addressed the need for stan-dards in labeling the various components ofa genome sequence. “I often run into peoplefrom the sequencing end who assume thatwhat they see on the computer is right whenthey look at annotation,” she said before

explaining the process and the problemsinvolved.

Land echoed Chain’s comment from thefirst day of the conference on the steadilyincreasing list of genomes and sequencesbeing made available. There’s a movementto make the annotation process automatedin order to speed up the work and make itmore consistent. Unlike manual review, shesaid, computers will always interpret thedata in the same way. And yet “consistentdoesn’t always mean better,” Land noted.“Sometimes it’s consistently wrong too.”

There are a number of genome databas-es out there that serve as references forannotation work, Land said, and the infor-mation in them is based on observationsmade by other people. Each of these tools,however, was created with a specific pur-pose in mind, which is why the data provid-ed by each might not always be useful toresearchers. In that sense, she added, thequality of the annotation can become

dependent on a person’s point of view,which is in turn defined by their knowledgeand experience.

All of these variations led to Land’s callfor annotation standards, which she didn’tthink was as impossible a process as Chainhad made it out to be during his talk.

“We want a method of assessing anddesignation quality as currently all annota-tion is deemed uniform in quality,” she said.“We want a standard vocabulary for whenyou’re talking about the same thing.” An“annotation quality sequence,” she said inclosing, deserves annotation of compara-ble quality.

Patrick Chain’s talk can be viewed onlineat http://www.scivee.tv/node/11406.Miriam Land’s talk can be viewed online athttp://www.scivee.tv/node/11424. Othertalks from the “Sequencing, Finishing,Analysis in the Future” conference can befound online at http://www.scivee.tv/node/11378.

Coming to Terms in Santa Fe cont. from page 2

Scientists studying climate changeoften look for organisms that can serveas the local version of the canary in acoal mine. Such sentinels helpresearchers identify warning signs of envi-ronmental changes that are likely to upsetthe balance of the ecosystem. In theArctic region, the scientists watch thepolar bears. In the oceans, however, theymight be watching something much, muchsmaller.

An algal genus known as Micromonasthrives in waters ranging from the equatorto the poles and plays a key role in theglobal carbon cycle. These microorgan-isms lend marine scientistsinsight on how climate changeand rising ocean tempera-tures affect marine ecosys-tems and which organismsmight become major playersin the global carbon cycle.

Approximately a 50th ofthe width of a human hair,Micromonas could giveOlympian Michael Phelps arun for his gold medals, beingable to move through waterat the rate of 50 bodylengths per second. The tinymarine microorganism canalso move toward the sun-light that powers it, much likesunflowers constantly keeptheir faces turned toward thelight throughout the day.

The DOE JGI sequencedgenomes of two Micromonassamples being studied byAlexandra Z. Worden ofMonterey Bay AquariumResearch Institute (MBARI).Taken from opposite ends ofthe world — the South Pacificand the English Channel —

approximately 10,000 genes were identi-fied in each isolate.

DOE scientists plan to use the geneticinformation to develop algal biofuels,among other things. The algal sequencescould also provide researchers with a lookat the encoded evolutionary record of howplants moved from the water to land.

“Genome sequencing of Micromonasand the subsequent comparative analysiswith other algae previously sequenced byDOE JGI and Genoscope [France], haveproven immensely powerful for elucidatingthe basic ‘toolkit’ of genes integral not

only to the effective carbon cycling capa-bilities of green algae, but to those theyhave in common with land plants,” saidDOE JGI Director Eddy Rubin.

But the genomic results also revealedthat the algae weren’t as similar to eachother as the researchers had previouslythought.

“These two picoeukaryotes, often con-sidered to be the same species, onlyshare about 90 percent of their genes,”said Worden. To put this in perspective,humans and some primates have about98 percent genes in common.

The information suggests the algae ineach region may thus reactdifferently to coming environ-mental changes.

“By understanding whichgenes a specific strainemploys under certain condi-tions, we gain a view intothe factors that influence thesuccess of one group overanother,” Worden said. “Wemay then be able to developmodels that could moreeffectively predict a range ofpossible future scenarios,that will result from currentclimate change.”

Worden thinks, for exam-ple, that the algae wouldadapt more easily than othermarine species because ithas already shown that itcan thrive in a variety of tem-peratures.

Only five other singlecelled marine microorgan-isms had had their genomessequenced prior to the publi-cation of the work, whichappeared in the April 10issue of the journal Science.

Ocean Sentinels for Climate Change

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10 / the PRIMERSummer 2009 Vol. 6, Issue 1

A 3D reconstruction of an electron tomograph-ic slice (0.5 microns thick) of one of the small-est known eukaryotic algae, Micromonas: A.Z.Worden, T. Deerinck, M. Terada, J. Obiyashiand M. Ellisman (MBARI and NCMIR). Background: Flavio Robles (LBNL).

the PRIMER / 11Summer 2009 Vol. 6, Issue 1

Kyrpides on Microbial Genomics

CSP 2010 Selections

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the list of microbial genomes for potentialsequencing has been limited to theapproximately one percent of organismsthat can be cultured in the lab and theirDNA then extracted. He sees a way for-ward using single-cell genomics — a tech-nique now being pursued in earnest byDOE JGI researcher Tanja Woyke and hercolleagues — in partnership with environ-mental metagenomics to provide a moreholistic understanding of a communityand its individual members.

Kyrpides also suggests several innova-tive approaches for easing the data pro-cessing bottleneck accompanying theexponential increase in genomic data. All-versus-all gene comparisons — previously

a common practice — will become infeasi-ble. To reduce the size of the datasets, heproposes a proxy approach in which oneprotein from each protein family or onespecies from each genus represents thegroup. Taking this one step farther, all thegenes from all the sequenced strains in aspecies — the pan-genome for that species— would constitute the genome repre-senting that species for gene comparisons.

Echoing other researchers, most notablyDOE JGI’s Patrick Chain and Miriam Landduring the recent “Sequencing, Finishing,Analysis in the Future” Conference, Kyrpidescalls for the development of genomeannotation standards and their adoptionby sequencing centers around the world

— a necessity formeaningful genomecomparisons.

With new tools inhand and internation-al initiatives forincreased collabora-tion underway, the field of microbialgenomics is poised for a decade of excit-ing advances. Sharing his vision for thefuture, Kyrpides observes: “To exploreand seek to understand how the Earthbreaths, grows, evolves, renews, and sus-tains life — all essentially the work of themicrobial world — is the great adventurenow beckoning to us. Microbial genomicspaves the way forward.”

gut samples taken from 11 insect speciesendemic to New Zealand and 3 termitespecies from Australia that potentiallyhave novel wood-degrading enzymes.

Another metagenome project involvesthe desert locust (Schistocerca gregaria),swarms of which spread over up to 20percent of the world’s land mass andaffect the livelihoods of up to 10 percentof the world’s population just by consum-ing the equivalent of their body mass daily.DOE JGI researcher Falk Warnecke pro-posed sequencing the insect’s gut walland the microbial community inside, inpart to find out if the locust or the microbesshould get the credit for degrading ligno-cellulose during these meals.

Many sequencing projects focus onbreaking down plant cell walls to get moreenergy out of biofuel feedstocks. Twoinvolving fungi come from Daniel Cullen, U.S.Forest Service, Forest Products Laboratory,who proposed sequencing a lignin-degradingwhite rot fungus known as Phlebiopsisgigantea, and Harvard University’s Anne

Pringle, whose proposal focuses on thecommon Amanita thiersii which breaksdown cellulose.

Other approved projects focus on thefermentation process of alcohol production.David Mills from the University of California,Davis and Trevor Phister from North CarolinaState University both proposed studyingbacterial contaminants that destroy alcoholsand reduce biofuel production efficiencies:the bacteria that turns wine into vinegar(Acetobacter aceti) and the bacteria respon-sible for the distinctive “horse-sweat taint”in contaminated wine (Dekkera bruxellensis).

The DOE JGI is also using DNA sequen-cing for projects related to carbon cyclingand biogeochemistry. A project from MarkWaldrop of the U.S. Geological Survey looksat microbes trapped in the Alaskan perma-frost, a carbon sink threatened by globalwarming. The samples to be sequencedcome from the USGS’ Yukon River Basinproject, where geoscientists have been study-ing the region’s response to climate changesince 2001.

Another project related to the carboncycle comes from Ramunas Stepanauskasat the Bigelow Laboratory for OceanSciences in Maine and involves microbialgenomes collected from the Atlantic Oceannear South America. Stepanauskas intendsto use single cell genome sequencing tech-niques, building on previous collaborationswith DOE JGI research scientist Tanja Woykeand her colleagues (see story on page 5).

One sequencing project is led byVolodymyr Dvornyk from the University ofHong Kong and involves cyanobacterial

cont. from page 1

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P. gigantea found fruiting on a red pine login a northern Minnesota forest, where thefungus, an aggressive saprophyte, is com-monly found on downed logs or cut timber.Photo by: Robert A. Blanchette, Professor,University of Minnesota

cont. on page 12

12 / the PRIMERSummer 2009 Vol. 6, Issue 1

22010SAVE THE SAVE THE DATE!DATE! MARCH 24-26WALNUT CREEK, CA

Contact The PrimerDavid Gilbert, Editor / DEGilbert@lbl.gov / (925) 296-5643

CSO 16843

samples of Nostoc linckia collected fromopposite faces of a valley known as Evolu-tion Canyon north of Tel Aviv, Israel. Thesouth side of the valley receives 800 per-cent more solar radiation than the northside of the valley, so the samples offerresearchers an opportunity to learn how

long-term stress affects the evolution ofthe bacterial genome.

Among approved projects that focus onmicrobes that can help clean up the envi-ronment is one from Gillian Lewis at theUniversity of Auckland in New Zealand.She proposed sequencing a novel isolate

of a freshwater bacterium from theSiderocapsaceae family that can removemanganese contaminants.

For the complete list of CSP 2010sequencing projects, see:http://www.jgi.doe.gov/sequencing/cspseqplans2010.html

CSP 2010 Selections cont. from page 11

Part of DOE JGI's strong presence at the at the 4thSequencing, Finishing, Analysis in the Future Meetingheld in Santa Fe, NM May 27-29. Left to Right: StephanTrong, Steve Lowry, Kurt LaButti, Alicia Clum, SusanLucas, Alla Lapidus and Hui Sun.

Matthias Hess, DOE JGIpostdoctoral fellow pre-sented the work on min-ing the metatranscrip-tome of the cow rumenmicrobiota for feedstock-targeted glycosyl hydro-lases, at the 31st AnnualSociety of IndustrialMicrobiology'sSymposium on Fuels andChemicals May 4 in SanFrancisco.

Twenty-three feet, four and a quarterinches and counting...At a recentgathering of poplar researchers atthe base of DOE JGI’s sentinelPopulus trichocarpa Jerry Tuskan(arms folded, above the helix), pre-dicted at least another six-footgrowth spurt by year's end.

U.S. House of Representatives Committee onScience and Technology staffers (left to right)Adam Rosenberg, Margaret Caravelli, JettaWong and Elizabeth Chapel, visited DOE JGI fora briefing on May 28.