Genome Technology

Contents | Zoom in | Zoom out For navigation instructions please click here Search Issue | Next Page This month in Genome Technology

Also inside this month’s Genome Technology • STRUCTURAL VARIATION The latest findings and approaches to understanding copy number variation regions and what they mean.

• METABOLOMICS Work is underway to improve the technology and analytical tools needed to understand phenotype.

• CASE STUDY Researchers aim for molecular diagnostics to decipher the mystery of metastatic carcinomas of unknown primary.

• CLINICAL Q&A: Gurvaneet Randhawa The genomics advisor opines on evidence- based medicine.

LOG ON NOW AT WWW.GENOME-TECHNOLOGY.COM RECENT RESULTS FROM GT POLLS Explore all of our exclusive new resources for molecular biologists: How do you keep up with research in your field? THE DAILY SCAN — Our editors’ daily picks of what’s worth reading on the Web I rely on PubMed. What else THE FORUM — Where scientists can inquire and comment on research, tools, is there? ...... 68% and other topics I go online, to community-based sites PODCASTS — Hear the full comments – in their own voices – of researchers like FriendFeed and blogs...... 10% interviewed in Genome Technology Magazine I use referrals from other scientists...... 4% POLLS — Make your opinions known on topics from the sublime to the ridiculous I stick to the basics: Nature and ARCHIVE — Magazine subscribers can read every word of every article we Science...... 18% have ever published

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SEPTEMBER 2008 THE ARTOF SPLICING Scientists work to unravel alternative splicing

Copy Number Variation Advances in Metabolomics Informatics for Glycomics

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SEPTEMBER 2008 Contents

“We’ve long thought that the expression of different splice isoforms probably underlies a lot of the differential effects of drugs in different pathways.” Diane Lipscombe, Page 41

Metabolomics Structural Alternative Taming Metabolites variation splicing Metabolomics studies inch scientists ever closer Counting on A Complexity that to understanding phenotype. Copy Number Goes Beyond Genes But to really make progress, Research into copy number Most genes are subject pioneers are working on variation is highlighting just to alternative splicing, but improving the technology how complex genomic differ- it’s still early days in under- and analytical tools ences are. A glimpse at the standing the phenomenon of the field. latest findings and approaches across pathways or on a BY CIARA CURTIN to understanding CNV regions genome-wide scale. A look and what they mean. at some pioneers in the field BY MEREDITH SALISBURY and the technologies they’ve commandeered to make sense of it. 31 BY JEANENE SWANSON 37 40 On The Cover 40 The Art of Splicing 37 Copy Number Variation 31 Advances in Metabolomics 25 Informatics for Glycomics

COVER AND TOP RIGHT PHOTOS: SAM RILEY SEPTEMBER 2008 GENOME TECHNOLOGY 3

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SEPTEMBER 2008 Contents

10 28 61 Markers In every issue Upstream GWAS ...... 10 PRIMER ...... 7 SEQUENCING...... 49 NHGRI issues $31M to The simple life? Not for us Barcode of Life project follow-up SNP research leans on sequencing WHERE ARE THEY NOW?...... 9 SAMPLE PREP...... 11 ImmunoPCR, microarray PROTEOMICS ...... 51 Marziali’s tech for DNA diagnostics, and a whole Satoris to launch purification tackles the new breed of institute Alzheimer’s test worst of contamination ZEITGEIST ...... 18 RNA INTERFERENCE ...... 53 GENE EXPRESSION...... 12 Something to talk about Tacere evaluates new Cancer consortium traces drug opportunities signature for lung cancer CAREERS ...... 20 recurrence Savvy strategies in a BIOINFORMATICS ...... 55 budget crunch Scientists debate publishing MICROFLUIDICS...... 13 future RainDance readies to ship INFORMATICS INSIDER ...... 23 early-access version of Homology: genealogy for genes MICROARRAYS...... 57 droplet platform For WTCCC, Agilent will BRUTE FORCE ...... 25 provide custom chips SUPERCOMPUTING ...... 16 Sweet time for informatics New processor architecture holds UNDER ONE ROOF ...... 28 promise for protein, gene studies Downstream Starting from scratch BOOK REVIEW ...... 16 PHARMACOGENOMICS...... 59 UPCOMING EVENTS...... 62 The Allen Reference Atlas: Pathwork first to net FDA OK for CUP Dx A Digital Color Brain Atlas of LAB REUNION ...... 64 the C57Black/6J Male Mouse A man of many hats CASE STUDY ...... 60 Diagnosing the unknown CLINICAL LABS...... 17 BLUNT END ...... 66 AACC annual meeting covers Q&A...... 61 genomics, proteomics, and Too much information diagnostics

GENOME TECHNOLOGY PODCASTS: A recent edition covered cloud computing, Esther Dyson, proteomics standards, and more. www.genome-technology.com

SEPTEMBER 2008 GENOME TECHNOLOGY 5

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FROM THE EDITOR Primer

ISSUE NO. 84

125 Maiden Lane, Second Floor New York, NY 10038 The Simple Life? Not for Us Tel +1 212 269 4747 Fax +1 212 269 3686 GENOME-TECHNOLOGY.COM______EDITORIAL DIRECTOR n looking over the pages of this issue of Genome Technology, it Meredith W. Salisbury occurs to me that there’s a theme here — however unintentional [email protected] SENIOR EDITOR it may have been when we started. Many of the articles this month Ciara Curtin [email protected] revolve around the concept of complexity. SENIOR WRITER Remember all those years ago when Incyte boasted about hav- Jeanene Swanson [email protected] I ing 100,000 human genes in its database? It was quite a disappoint- REPORTER ment when the community homed in on the real number of human Matthew Dublin [email protected] ART DIRECTOR genes, leaving our egos just a bit bruised as we wondered how we could Paul J. Schindel [email protected] be such delightfully complex creatures when we had the same gene DESIGN & PRODUCTION count as a mouse (or, for that matter, Arabidopsis). Lois Savander • John Burrows As GT readers have known for a long time, there’s a GENOMEWEB DAILY NEWS lot more than gene count factoring into genetic diver- Bernadette Toner, Editorial Director, News [email protected] sity. Our cover story delves into the world of alterna- Ed Winnick, Managing Editor tive splicing, a genomic phenomenon that allows us [email protected] to be economical in number of genes but without Matt Jones, Reporter [email protected] skimping on the products they encode. As Jeanene Andrea Anderson, Reporter [email protected]______Swanson reports, scientists are using a range of tech- GENOMEWEB NEWSLETTERS nologies to study why and how alternate splicing Kirell Lakhman, News Editor [email protected] takes place — as well as the effect it has on organism Justin Petrone, BioArray News Editor development. Research in this field has led to a bet- [email protected] Vivien Marx, BioInform Editor ter understanding of diseases, particularly in neurodegenerative condi- [email protected]______tions that have proven difficult to make sense of with other approaches. Alex Philippidis, BioRegion News Editor [email protected]______While splicing adds a significant layer of complexity to genomic stud- Ben Butkus, Biotech Transfer Week Editor ies, so too does copy number variation, the focus of a feature story in [email protected] Charlotte LoBuono, Cell-Based Assay News Editor this issue. While we may have just 20,000 (ish) genes, we’re sneaky [email protected]______with them, packing our genomes with copies of the same genes. Some- Julia Karow, In Sequence Editor [email protected] times they’re inverted or changed ever so slightly, as if our genome was Turna Ray, Pharmacogenomics Reporter Editor [email protected] afraid of getting caught stacking the deck. In our article on CNV, we Tony Fong, ProteoMonitor Editor checked in with scientists leading the field to find out more about [email protected]______Doug Macron, RNAi News Editor increasing use of variation studies in model organisms, the tools [email protected] needed to accurately and comprehensively find gene copies, and how CHAIRMAN AND PUBLISHER the mechanism has helped scientists establish links to disease. One Dennis P.Waters, PhD [email protected] expert we interviewed, Harvard’s Charles Lee, went so far as to pre- ADVERTISING/SALES dict that a clear link of cancer predisposition with copy number vari- Judy Block, Associate Publisher [email protected] +1 212 651 5629 ation is “right around the corner.” Allan Nixon, Director of Business Development Of course, complexity goes way beyond the genomic level. Ciara [email protected] +1 212 651 5623 David Samuels, Subscriptions and Curtin reports on the growing field of metabolomics. Her feature story Site License Manager describes the advances researchers have made in the detection and [email protected]______OPERATIONS AND FINANCE deconvolution of metabolites, while noting that they’re still grappling Greg Anderson, Director with establishing more comprehensive databases. In his Brute Force [email protected]______Philip Borowiecki, Associate column, Matt Dublin considers the nascent glycomics space, focusing [email protected] on the informatics at play there as well as on the community’s attempt to implement standards early in the game.

Copyright ©2008 GenomeWeb LLC. THE GENOMEWEB INTELLIGENCE NETWORK No portion of this publication may be reproduced in whole or in part without written permission from GenomeWeb. Meredith W. Salisbury, Editor

SEPTEMBER 2008 GENOME TECHNOLOGY 7

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WHERE ARE THEY NOW? Update

>GT ONLINE www.genome-technology.com GT ONLINE POLL RESULTS: Grants are tougher to ImmunoPCR, Microarray Diagnostics, come by; what's the best approach to still get one? and a Whole New Breed of Institute 17% SUBMIT AT LEAST TRIPLE THE USUAL NUMBER OF year ago in Genome Technology, a feature GRANT APPLICATIONS story checked into immunoPCR as an up- and-coming diagnostic tool. Only 15 years old, the technique couples an anti- 2% body-based detection assay with real-time ASK FOR LESS MONEY AND A PCR, allowing for the detection of proteins and YOU'RE MORE LIKELY TO GET FUNDED viruses in very small concentrations. At the time, TATAA’s Mikael Kubista was tweaking his PSA test to use immunoPCR instead of ELISA, and just one com- 43% pany — Chimera Biotec — was selling customized IT'S NOT ABOUT MORE GRANT immunoPCR kits. While the technique continues to SEPTEMBER 2007 APPS, BUT MORE TARGETED make headway in the lab — it’s used in drug discov- ONES; SPEND MORE TIME ery, biomarker detection, and as a clinical lab tool — CRAFTING YOUR APPLICATIONS tricky conjugation protocols have made it difficult to standardize and move beyond the bench. 37% Last year’s cover story took an eagle’s eye view of the PARTNER WITH SOMEONE ELSE microarray diagnostics arena, reviewing the main AND SUBMIT A JOINT PROPOSAL players and the chip-based assays they were developing. Roche’s AmpliChip CYP450 and Agendia’s >NEED HELP? MammaPrint were the first array-based diagnostic CHANGE OF ADDRESS — assays to gain FDA approval. Others were hard at SEPTEMBER 2003 To change your address or work creating their own tests: Heidi Rehm at Harvard manage your subscription to Genome Technology, the Daily -Partners Center for Genetics and Genomics was developing tests for hearing Scan, or any Genome-Web publi- loss and for hypertrophic cardiomyopathy, and Emory’s Madhuri Hegde was cation, sign in to www.genome-______developing one for X-linked muscular dystrophy. In July of this year, Path- technology.com______and click on work Diagnostics’ Tissue of Origin test became the second in vitro diagnostic My Account, or e-mail us at multivariate index assay to be cleared by the FDA. [email protected]. In September 2003, our cover story delved into the rise of the systems biol- FREE SUBSCRIPTION — Genome Technology is free to ogy community by tracking seven pack leaders. The story looked at the lead- active researchers in the life sci- ership, structure, and goals of these new interdisciplinary biology centers, ences. To subscribe, log on to which included Stanford’s Bio-X Program for Bioengineering, Biomedicine, and www.genome-technology.com Biosciences; MIT’s Computational and Systems Biology Initiative; Princeton’s and fill out a 30-second form. Lewis-Sigler Institute for Integrative Genomics; Duke’s Institute for Genome REPRINTS — For permission Sciences and Policy; the University of Michigan’s Life Sciences Institute; the to reproduce material from Genome Technology, or to California Institute for Quantitative Biomedical Research; and the Cornell Life order offset-printed or Web- Sciences Initiative. These have heralded a wave of similar systems biology insti- ready PDF reprints, write to tutes focused on meshing different disciplines for large-scale, collaborative [email protected]______research. (The trend has become so popular that GT has since created a spe- or call +1.212.651.5632. cial column that profiles a new systems biology center each month.) Some of ARCHIVE ACCESS — the multimillion dollar institutes that have graced our pages this year include Subscribers can search through the complete GT archives by Barcelona’s Centre for Genomic Regulation, the Michael Smith Genome Sci- issue or keyword. Simply log ences Centre in Canada, and the Burnham Institute in California. in on the website. — Jeanene Swanson

SEPTEMBER 2008 GENOME TECHNOLOGY 9

Markers NEWS

GWAS: NHGRI Issues $31M to >SHORT READS Follow-Up SNP Research

Last month, Alan Guttmacher took on his new role as acting director of NHGRI after Francis lush with $31 million in of America. Her Collins officially stepped down. funding from the National population is one- Guttmacher is a pediatrician Human Genome Research third each Mexi- and medical geneticist, and Institute, four researchers can-American, has been deputy director of will be following up on African-American, the genome institute for F putative disease-related SNPs iden- and European- more than five years. tified through genome-wide associ- American. Most of ation studies. These scientists will the GWAS SNP The US Departments of be examining the prevalence of discoveries so far Energy and Agriculture common disease SNPs in different, have been con- DANA CRAWFORD will give nearly $11 million already existing epidemiological ducted in populations of European over three years to fund 10 cohorts to see if they are still linked descent, and looking in her cohort genomics research programs to disease in general populations. could show if the SNP-disease that can help develop bio- “If you think about a typical association holds true in a more energy feedstocks for use in genome-wide association study as diverse population. cellulosic biofuels. Recipients looking at many genetic variants in Not all cohorts under investigation include the University of relation to one or a few health out- are cross-sectional; others have fol- Georgia, Penn State, Michigan comes, in this follow-up program, lowed participants for decades. Fred State, Oregon State, and the we’re looking at one or a few Hutchinson Cancer Research Center’s University of Massachusetts, genetic variants in relation to many Charles Kooperberg will be studying among others. health-related outcomes,” says SNPs in the Women’s Health Initia- Lucia Hindorff, program director at tive population, which has followed In response to the growing NHGRI. 160,000 women since 1991. “We number of so-called “minimum GWAS research uncovers genes have a wealth of epidemiological information” checklists for associated with disease, but does so data. We have loads of clinical out- high-throughput biology in a population predisposed to find comes data,” he says. experiments, an effort known such SNPs, even in case-controlled Just which SNPs and what epi- as Minimum Information studies. “Which is great for discov- demiological data will be the focus, about a Biomedical or Crawford and Kooper- Biological Investigation is “We hope to be able to fill in berg don’t yet know, but attempting to provide a set of the common diseases are guidelines for standardization gaps about what’s known.” likely to include diabetes, groups to ensure inter- heart disease, and cancer. operability and prevent ery,” says Vanderbilt University’s Crawford and Kooperberg, along duplication of effort. Dana Crawford, who received $7 with the Gerardo Heiss at the Uni- million of this grant. “But when versity of North Carolina, Chapel Affymetrix acquired you’re trying to describe what the Hill, and Loïc le Marchand at the True Materials, a firm with SNP looks like in the general pop- University of Hawaii Research Can- microparticle technology for ulation — we don’t have any data cer Center will act as a consortium use in diagnostic applications, on that yet.” and soon will meet to prioritize a for about $25 million in cash. The cohort that Crawford will be list of SNPs. “We hope to be able to Randy True, founder of the working with is from a long-run- fill in gaps about what’s known San Francisco startup, joined ning Centers for Disease Control about genetic variants in these Affy as vice president of and Prevention cross-sectional populations with this program,” research and development. study that, as she says, takes a slice says Hindorff. — Ciara Curtin

10______WWW.GENOME-TECHNOLOGY.COM SEPTEMBER 2008

NEWS Markers

Sample Prep: Marziali’s Tech For DNA Purification Tackles >SHORT READS

Illumina completed its The Worst of Contamination acquisition of Avantome, a t’s not every day you see biol- was able to deliver privately held developer of ogists get really excited about a purified micro- low-cost, long read-length — of all things — sample gram of DNA — sequencing technology, for prep. But if Andre Marziali has enough to get the $25 million in up-front any say in it, you can expect project going. payments and contingent I payments of as much as that kind of enthusiasm to become Currently, downright commonplace. Marziali is work- $35 million. The startup was Marziali is the brain behind ing on a second- co-founded by Stanford’s SCODA, a nucleic acid separation generation proto- Mostafa Ronaghi, who joins technology that relies on special type, which would ANDRE MARZIALI Illumina as senior vice presi- electrical fields to purify DNA or speed up the purification process dent and chief technical RNA out of even highly contami- from a few hours to as little as five officer. Separately, Illumina nated samples. The technology has minutes. Boreal is hoping to get a announced that it named just been exclusively licensed from beta version of that instrument out Stephen Pentoney to the post Marziali’s research home at the to customers by early next year. of VP for assay and reagent University of British Columbia to The cost will be comparable to development in life sciences. his startup, Boreal Genomics, for similar electophoretic products, commercialization. Marziali says: “In the future we Joanne Sun is now director By the start of this year, Marziali imagine an instrument priced of protein analytics and high- already had prototype instruments below $10,000.” throughput purification at the out in the field, where researchers The tool, and the concept behind antibody discovery company were getting acquainted with the it, has proven so popular that Adimab. She formerly worked gel-based method that uses focus- Marziali finds himself in the in clinical trials for Adnexus ing electrical fields, which nucleic unusual situation of having to Therapeutics and in pre-clinical “turn investors away.” Tom development at Abbott. “When we first started Willis, who chairs Boreal’s board of directors, helped A group of scientists led by applying this to DNA, we Marziali get the company NHGRI’s Colleen McBride off the ground and work published a commentary in didn’t know how selective it out a business model rely- Nature Genetics calling for an was going to be. It’s just ing on as little venture cap- increased emphasis on trans- ital as possible. lational research to make sure played out beautifully.” The technology, which that personalized genomics can take any number of delivers on its promise. They acids respond to but contaminants sample types from soil to blood to contend that regulators and don’t. The fields concentrate the milk, is broadly applicable in members of the biomedical DNA or RNA into a compact unit at forensics, agriculture, genomics, communities need a better the center of the gel, while all other and throughout the life sciences. understanding of how informa- molecules are spun out to the Marziali says he stumbled across tion from genetic tests is used periphery of the gel. In a metage- the idea for SCODA quite by acci- in the clinic, how useful those nomics project with Rob Holt, dent. “When we first started tests are, and how genetic Marziali describes taking a soil applying this to DNA,” he says, “we knowledge is viewed and used sample that no one else had been didn’t know how selective it was by patients before genetic able to extract DNA from. Using the going to be. It’s just played out technologies find their way SCODA technology, Marziali’s team beautifully.” — Meredith Salisbury into mainstream use.

SEPTEMBER 2008 GENOME TECHNOLOGY 11

Markers NEWS

Gene Expression: Cancer >SHORT READS Consortium Traces Signature

An international team of researchers from Germany, For Lung Cancer Recurrence the US, Croatia, and Finland arly-stage lung cancer same conditions used the Roche 454 sequenc- patients are usually for isolating sam- ing platform to sequence the treated surgically and sent ples, the same cri- Neanderthal mitochondrial home, but 25 percent to teria for inclusion, genome to about 35 times 30 percent of those people and reagents from coverage. The publication E will have their cancer recur. the same produc- in Cell was based on DNA Researchers from cancer institutes tion lot. Samples isolated from a bone more in the United States and Canada are from two of those than 38,000 years old paving the way to develop gene sites became the that was discovered in expression panels to predict which training set, and DAVID BEER Vindija Cave in Croatia lung cancer patients will relapse. “If researchers from the four institu- in 1980. you know who these high-risk tions independently used those patients are, then you would samples to build predictive models. The Washington University potentially be able to offer them “The models that we tested were School of Medicine in St. Louis additional therapy,” says David very, very broad from one gene to hired Barry Sleckman as direc- Beer, a professor of surgery and thousands of genes, and they all tor of the Division of Genomic radiation oncology at the University encompassed different approaches,” Medicine. Sleckman, who of Michigan. Beer says. joined the school 10 years To develop a gene expression Of the four panels developed, two ago as an assistant professor panel that can predict which lung were able to predict prognosis in of pathology and immunology, cancer patients have a poorer prog- the independent, blinded test sets. studies DNA repair and devel- nosis than others, researchers need Furthermore, when the gene opment of the early immune a lot of samples to analyze for mark- expression panels were used in con- system. ers — more than they typically have junction with the clinical data, access to. To manage that, Michi- they worked even better. “That BioNanomatrix received a gan, H. Lee Moffitt, Memorial meant that putting the two together $399,020 grant from NHGRI Sloan-Kettering, and Dana-Farber is more powerful than using just to continue development of formed a consortium to track one alone,” Beer says. The results its nanoscale imaging platform are reported in the August for haplotyping and gene “We really need to look issue of Nature Medicine. mapping in a massively While not yet ready for parallel format. This is the at a larger number of the clinic, these results fourth grant the company indicate that “there is infor- has won from NIH to work tumors.” mation in the genes that on the technology. can tell you about the down a gene signature for lung can- behavior of tumors,” says Beer. He The Broad Institute of MIT cer prognosis. “We really need to and his colleagues are now working and Harvard released its look at a larger number of tumors,” to determine whether the morpho- Integrative Genomics Viewer, says Beer. “We decided to basically logical heterogeneity of lung cancer an informatics tool that allows pool our resources and put all of tumors has a molecular basis. “It’s scientists to visualize genomic our tumors together.” possible there’s a lot of different ways data. The tool’s zooming and In this three-year study, the that lung cancer becomes a cancer,” panning abilities make it feel researchers collected 442 lung can- he says. “Genomically it’s a more like Google Maps, according cer samples from different treat- difficult problem to unravel that.” to Broad scientists. ment sites. Each location used the — Ciara Curtin

12______WWW.GENOME-TECHNOLOGY.COM SEPTEMBER 2008

NEWS Markers

Microfluidics: RainDance

Readies to Ship Early-Access >SHORT READS Celera named Jean Amos Version of Droplet Platform Wilson as president of labora- f all goes according to plan, recent approaches tory operations at its Berkeley RainDance Technologies aims to of doing this with HeartLab subsidiary. She was get its droplet-based microflu- arrays or in solu- previously senior director of idics tool into the hands of tion “create bias” genetic services at Sequenom. early-access researchers this but that “using a I Researchers from the fall, says Steve Becker, vice presi- very well-refer- dent of commercial operations. enced PCR” would University of Toronto used Becker says that the main chal- lead to a far less high-density oligonucleotide lenges in high-throughput biology biased product. arrays to look at CNV frequency today are miniaturization, automa- They’ll work to STEVE BECKER in the general population and tion, and multiplexing — all issues show evidence of that in a partner- in families with Li-Fraumeni, a that he believes the company’s Rain- ship with Scripps, where scientists syndrome predisposing individ- Storm technology addresses head- will use the RainStorm technology uals to cancer, finding that peo- on. “Doing research in droplets for targeted resequencing. ple with more CNVs in their allows people to go back to what I’ll The droplets can be used to per- genomes may also be at call simplicity,” he says. “Each form “just about every general lab increased risk for cancer. The droplet is the functional equivalent application,” Becker says; they’re group, led by senior author of a test tube or a well.” The droplets thermostable and biocompatible, as David Malkin, published in the can be processed at speeds of 3,000 well as consistent at the picoliter Proceedings of the National per second, or more than 10 million size. “We’re able to pack many of Academy of Sciences. samples in an hour. “Having that these droplets next to each other and kind of throughput allows you to do they will not coalesce” — unless, Larry Wellman joins OpGen ultimately single-cell or single- that is, you want them to. The as HR veep. He was previously molecule [experiments],” he adds. droplets can be merged on demand, in the same position at Digene, The technology will first be or sorted “using [a] soluble fluores- now part of Qiagen. launched for the targeted rese- cent protein marker and a laser,” quencing market. Becker says that Becker adds. BioTrove hired Derek Potter while next-generation sequencing is RainDance was founded in 2004, as director of European busi- “growing at an unprecedented rate,” in part by 454 Life Sciences’ ness operations. Potter was Jonathan Rothberg, who previously European sales “Doing research in droplets is now chairman of the manager for Applied board. The company is Biosystems, and he was allows people to go back to currently developing involved in establishing and manufacturing the Fluidigm’s European what I’ll call simplicity.” droplet platform that operations. will go out to cus- scientists have not yet had an effi- tomers. According to Becker, sci- OpGen, a single-molecule cient way to perform genome entists will send RainDance their analysis company based in enrichment for resequencing. Using list of loci of interest; the company Madison, Wis., will open a RainDance’s tool, scientists would will create a library of droplets facility in Gaithersburg, Md. have one primer pair per droplet for with the corresponding primers; The company says it expects as many regions as desired, and then and then the library will be to hire 80 people in the next they’d perform “good old-fashioned shipped to the customers for use in two years to staff the facility, PCR” in emulsions within those their labs. which will house R&D, service droplets. Becker contends that — Meredith Salisbury operations, and manufacturing.

SEPTEMBER 2008 GENOME TECHNOLOGY 13

Scientist, Top 10 Pharmaceutical Company

Associate Director, Clinical Programs, Pathwork Diagnostics

Executive Vice President, Operations International Neuroscience Network Foundation

Senior Research Associate, Bay Area Biotechnology Company

Associate Director, Cambridge, MA Pharma Company Steve Tirrell, Millennium Pharmaceuticals, Inc.

EXPRESSIONS of

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Markers NEWS

searches of this kind would take Supercomputing: New hours and hours to complete on a typical cluster or supercomputer. But with an algorithm specially Processor Architecture Holds ported to this multithreaded processing architecture, the same job Promise for Protein, Gene Studies takes mere seconds to complete, supercomputing archi- that many disparate sets says Bader. “These sorts tecture that first of complex data can be of problems have over- appeared in prototype digested at once, instead whelmed modest size form more than 10 years of each portion of data clusters, and if you start Aago has been given a being handled piece by adding processors to a new lease on life, thanks in part to piece. cluster, it takes longer a recent $4 million Department of David Bader, a com- and longer to run Defense grant issued to seed the puter scientist at Georgia because the communica- new Center for Adaptive Supercom- Tech, demonstrated the tion costs dominate,” he puting Software. The joint project architecture’s application DAVID BADER says. “This is really the teams up Pacific Northwest to biology by identifying proteins first architecture where you can National Laboratory and super- that, when knocked out, disrupt pose a biological hypothesis, test it computer maker Cray, as well as the cancer-causing networks in a out, and run it in short seconds or several institutions including particular cell. Bader and his team minutes versus hours to days, or Georgia Institute of Technology used a social networking algo- maybe never.” and Sandia National Laboratories. rithm to mine a huge collection of Bader and his colleagues believe The initiative aims to develop publicly available human pro- the concept could offer a lot to large- software that takes advantage of teome datasets. “This is similar to scale life science computing prob- the multithreaded processing finding important people in a lems. “I think as we gather more capabilities of Cray’s XMT super- social network, sometimes called genomics data we can use such a computer. Unlike traditional ‘connectors,’” Bader says. “Looking system to make scientific discover- supercomputer processing archi- for these proteins is like looking ies,” he says. “I would hope, looking tecture, where each processor gets for a needle in a haystack — and it at three to five years, that we keep a portion of memory for each cal- is usually computationally inten- investing in these novel types of culation in a piece-by-piece fash- sive that won’t work well on architecture and looking at the sci- ion, the new processors are each current [high-performance] entific results we can achieve, espe- capable of multiple, simultaneous machines, but this new architec- cially in the areas of solving data crunching and use a much ture is really designed for this type genomic problems and understand- larger pool of memory per proof problem.” ing of the genome.” cessing core. This design means Normally, multiple database — Matthew Dublin

gene expression database, the atlas Book Review includes coronal and sagittal sec- tions and color-coded brain struc- The Allen Reference Atlas: A Digital Color Brain Atlas of ture delineations. It comes with a the C57Black/6J Male Mouse, free CD-ROM, which can be used in by Hongwei Dong and the Allen conjuction with online graphical, Institute for Brain Science gene expression, and informatics tools. In this atlas, authors Hongwei Dong and the Allen Institute for Publisher: John Wiley & Sons, Inc. Brain Science have created a full- Publication date: January 28, 2008 color brain atlas of Nissl-stained adult male mice. To be used in com- ISBN: 978-0-470-05408-6 fresh-frozen tissue from young plement with the Allen Brain Atlas — Jeanene Swanson

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NEWS Markers

determine warfarin sensitivity. The Clinical labs: AACC Annual simple procedure consisted of signing a few consent forms, spit- ting into a tube, and then going to Meeting Covers Genomics, a designated website where the results were posted. Proteomics, and Diagnostics Finally, to put it all in perspective, t the American Associa- of Pennsylvania School of Medicine, veteran broadcast journalist and tion for Clinical Chem- spoke about the use of standard political commentator for ABC istry annual meeting held genomics and proteomics tools for News, Cokie Roberts, gave a lively in Washington, DC, at molecular diagnostics and high- plenary on the healthcare debate — Athe end of July, it was the throughput genotyping, especially what the US presidential election will changing role of clinical laboratory for cancer. While his talk focused bring in terms of the candidates’ medicine in improving healthcare on miRNAs, he touched on many ideas for healthcare reform. “It is def- that garnered the most attention. In up-and-coming research tools that initely going to be robustly debated one of the first plenary sessions, Roy could be utilized for functional in this election campaign,” she said. Vagelos, retired chairman and CEO genomics, including alternative “It’s a different debate from what it of Merck, talked about the changing RNA splice arrays, tissue micro- used to be.” She contended that this pharmaceutical industry and its arrays, cell transfection microar- election will see people demanding evolving role in providing healthcare. rays, and “next-next-gen” sequenc- healthcare reform, and the statistics One symposium followed up on that ing, which uses nanotechnology to are enough to explain why: from with a look at evidence-based medi- get down to the single-molecule 2001 to 2007, worker earnings in the cine, while another delved into the level. “Functional genomics challenges facing clinical laboratory has long way to go,” he said, testing in the developing world. citing headway made in “We need good ways of James Hughes, director of Emory proteomic technologies as looking at protein-protein University’s program in global infec- just the start. “We need tious diseases, took on some of the good ways of looking at interactions and sub-cellular challenges and opportunities in cre- protein-protein interactions ating diagnostics for the developing and sub-cellular localiza- localization to really world. Those included managing tion to really understand in understand ... what’s the global burden of infectious dis- this entire model, what’s ease, dealing with emerging micro- happening to the whole happening to the whole bial and vector-borne threats that genome context.” have cropped up in the past decade, A debate on whether war- genome context.” and implementing disease control farin testing was ready for efforts. Some of the most devastating primetime was also the subject of a US went up 18 percent, while the diseases globally in terms of mortal- full-day symposium, with a series of cost of health insurance premiums ity are HIV/AIDS, tuberculosis, and experts in the field giving both sup- increased by 78 percent, Roberts malaria, Hughes said, citing data porting and opposing arguments. said. More than 25 percent of people from a 2003 World Health Organiza- While some say using genetic tests say they have trouble paying for tion study. “What you don’t hear to determine warfarin dosing is healthcare and insurance and “with much is that, actually, the leading ready to lead the way for more serious illness, it makes it consider- infectious disease killer worldwide is pharmacogenomic tests, others say ably worse,” said Roberts. “For those lower respiratory tract infections, the data is not yet available and that who are fighting cancer, which now primarily pneumonia and influenza, testing could increase costs while is one in three of us, 25 percent … and that coming in third is diarrheal not offering any real benefits. said the disease had used up much or disease,” he added. Inside the exhibit hall, ParagonDx all of their savings. I think that the At the other end of the Walter E. paired with DNA Genotek to offer a pressure on the national govern- Washington Convention Center, warfarin demo to conference atten- ment to act is going to be very Donald Baldwin, director of the dees, including a test for CYP2C9 strong.” microarray facility at the University and VKORC1, the two variants that — Jeanene Swanson

SEPTEMBER 2008 GENOME TECHNOLOGY 17

Zeitgeist BLOGOSPHERE BRIEFS

Something to Talk About The blogosphere has been chattering about job prospects, George Church, women in science, and Charles Darwin. By Ciara Curtin

Saving Your Job Be Whatever You Like

With the uncertain economy, there have been rum- New York Times blogger John Tierney examines apply- blings about layoffs and takeovers. At In the Pipeline, ing Title IX, the law banning sexual discrimination in Derek Lowe has a bit of job news: he heard that there education, to the sciences. The National Science Foun- will be chemistry layoffs at Pfizer come the fall, and that dation, NASA, and the Department of Energy have a potential Roche takeover of Genentech has gotten the been looking for instances of sexual discrimination at latter’s employees to look elsewhere for gainful employ- universities receiving federal funds by examining lab ment. Lowe also doles out some advice on how to keep space and interviewing researchers. Critics worry this your job from being sent abroad: start generating ideas will lead to a quota system for women in science. A and doing more difficult chemistry. “You have to bring blogger at Adaptive Complexity writes that having something that can’t be purchased so easily overseas,” quotas “would certainly make women second-class he writes. citizens in science, because they could never be http://pipeline.corante.com judged on their own merit.” http://tierneylab.blogs.nytimes.com/ www.scientificblogging.com/adaptive_complexity/blog

The Summer of George Deleting Darwinism

George Church exploded onto the mainstream scene With anticipation mounting for the 150th anniversary recently. A profile in Wired shows the winding path that of Charles Darwin’s theory of natural selection (and his his career has taken. (That same article, as noted by the 200th birthday), the man himself takes center stage. A Genetic Genealogist, unmasked the Personal Genome blogger at 3 Quarks Daily links to videos of Richard Project’s tenth participant: Harvard psychologist Steven Dawkins discussing his hero. But it’s a suggestion of Pinker.) In an interview with Charlie Rose, Church says Olivia Judson’s that catches on; she wants to get rid of that people should initially have low expectations of the term “Darwinism.” The 3 Quarks Daily blog jumps personal genomics. Valleywag also notes that one of on the bandwagon, as do Evolgen, Larry Moran at Church’s many advisory roles is with 23andMe, and Sandwalk, and Mike the Mad Biologist. “Modern evo- that one of the co-founders of that company, Anne lutionary biology has gone way beyond Darwin’s origi- Wojcicki, is married to Sergey Brin of Google, which nal ideas and it’s no longer appropriate to describe the has backed Church’s PGP. modern ideas as ‘Darwinian,’” writes Moran. http://www.thegeneticgenealogist.com/ http://judson.blogs.nytimes.com/ http://valleywag.com/ http://3quarksdaily.blogs.com/ http://scienceblogs.com/evolgen http://sandwalk.blogspot.com/ http://scienceblogs.com/mikethemadbiologist/

18 WWW.GENOME-TECHNOLOGY.COM______SEPTEMBER 2008

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Careers PROFESSIONAL LIFE

Savvy Strategies in a Budget Crunch In a tight funding climate, what’s the best way to get a grant? Scientists are trying to increase their odds by submitting more proposals, adding senior PIs to their applications — and other choices that are just plain wrong. By Meredith Salisbury

ome people call it the Just as there are good seems like a no-brainer, “undoubling” — methods for writing a chances are, it’s working referring to the current grant application in gen- against you, says Joanne federal funding situa- eral, there are strategies to Tornow, acting director of Stion, which after sev- follow when submitting a the division of molecular eral years of budget increases has proposal in this kind of and cellular biosciences at now seen a dramatic fall-off thanks funding climate. the National Science Foun- to inflation and years of flat appro- The natural tendency of dation. Funding agencies priations. anyone looking at dwin- don’t want to cut the dollar The funding crunch has led to a dling odds is simple: place NORKA RUIZ BRAVO amount of the awards host of other problems: skyrocket- more bets. Review panels are inun- they’re giving, so they’re more likely ing numbers of grant applica- dated with proposals as scientists have to cut the number of awards granted. tions, plummeting success rates, taken to submitting significantly At NSF, Tornow says, if you submit increasing age of winning a first more applications than they nor- several proposals that appear related, grant, and more. mally would. While the strategy they’ll be evaluated together — and

The Scoop

Fewer proposals funding opportunity. It’s also just a good idea to get to Of course, while there’s a risk that someone else will know the people at the funding agencies. keep submitting extra grant proposals, getting anyone to Don’t play it safe stop the application spree has slim chances of success. In a funding crunch, scientists tend to cut off the more But NSF’s Joanne Tornow says that driving up the number radical ideas in favor of the safer, more run-of-the-mill of proposals can weaken your overall chances and that proposals. Tornow says that’s a mistake; her agency your worst, rather than your best, submission may have a looks for research projects that push the envelope. greater impact on reviewers. Collaborate for expertise Targeted applications If your proposal includes a type of research that you One way to see how well your research idea would fit in haven’t attempted before, it might help to find a collabora- with a program’s guidelines is to see what kind of projects tor who has expertise in that particular area. Just make it have been funded through that program in the past. Make clear in the application who will be responsible for what. use of agency grant listings, such as NIH’s CRISP, to see Don’t lean on established scientists what’s been successful before. Apply to the programs that Younger investigators fearing they won’t get grant fund- seem like a natural fit for your research concept, and skip ing may be tempted to add a more experienced scientist the ones that lean in a different direction. as a co-PI on the application. Norka Ruiz Bravo and Tornow Make contact agree that this can undermine your ultimate goal of estab- There’s no substitute for getting in touch with the pro- lishing yourself as an independent investigator. Agencies gram directors themselves. You’ll get the most up-to-date tend to have special opportunities for scientists early in information about the program and specific advice on the their careers, so first try your hand with those.

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the weakest of the batch may reduce those? If your proposal would be an Young investigators nervous about the overall enthusiasm of reviewers outlier from the types of projects funding tend to consider teaming for any of your proposals. that have been successful, focus up with a more established scientist A stronger approach, Tornow says, instead on finding one that’s a bet- to act as co-PI on the grant, figuring “is to be very targeted on the pro- ter fit. Avoid the trap of playing it it will improve their odds. While col- posal that you write.” To that end, do safe, though: Tornow says her laborations can be a great idea, lean- your homework: certain agencies agency looks for ideas “that are a ing on more experienced scientists fund certain kinds of research, and little bit more out there” — which can backfire. “I would advise some- the programs within those agencies means “you need to guard against one to be independent as quickly as are more specific still. With limited those tendencies to move toward possible,” says Ruiz Bravo. Even if the funding, each program director has the safe stuff.” grant idea is your own, you run the to make sure every choice aligns with Once you’ve found a program risk of being perceived as riding on the program’s priorities. From NSF’s you’re interested in, the best way to the other scientist’s coattails. perspective, “there is a need to build get the inside scoop is to contact the Still, teaming up is a good idea in portfolios and to think about where program director and ask questions. cases where you’re proposing to do our investment can have the greatest “The best thing that anybody can work you haven’t been trained to do, impact,” Tornow says. do is get on the phone and call us,” says Tornow. In such a situation, When you scope out a program says Norka Ruiz Bravo, director of find someone who has expertise in announcement, do some digging to the Office of Extramural Research at that area and add that person to the see what kinds of projects have NIH. If cold calling intimidates you, proposal. Even then, Tornow cau- been funded by that program (or a try e-mail — it’s unobtrusive and can tions, “it is going to be important for similar one) in the past. Does be a great way to ask a simple ques- it to be clear whose intellectual yours fit the scope and direction of tion or two. input is driving the project.” Next-Gen DNA Shearing Tune in High Definition Sample Reproducible ™ N=12 Preparation with Covaris Systems Avg=100bp CV =2.6% S2 and E210 systems TE buffer 100ul volume Lambda DNA (starting material)

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FRAN LEWITTER & GEORGE BELL Informatics Insider

Homology: Genealogy for Genes Homology may be a keystone of biology, but confidently identifying homologs is a challenge. Here, we go beyond basic sequence comparisons to look at better methods to assign homology.

he great power of people, we’re often asked useful. To assign pairs model systems in to identify the homolog of of homologs A and B molecular biology a human disease-causing across species, genome- has been apparent gene in another species, scale analyses often go at T ever since early whether that be mouse, least one step further than researchers used bacteria, yeast, zebrafish, yeast, or our Blast search above, worms, and flies to learn about the another model system. requiring that B is the human body. More recently, the Just about any molecular most similar protein to A power of comparative genomics biologist can now use and vice versa. If we find has been harnessing evolution to Blast to take a human pro- FRAN LEWITTER an orthology pair like help identify the most obviously tein to search a database this, we’re in good shape, important parts of our DNA by of, for example, zebrafish but do we want to further linking a piece of one genome to a proteins to identify the restrict our measure of corresponding piece of another most similar one. Is the similarity? What if we can genome. top hit the homolog we’re generate only a short local On a molecular level, both of these looking for? We can’t be alignment? What if a sim- approaches can require that we link sure, and this gets at the ilar analysis of gene genes that are homologous, i.e., crux of the definition; sequences is inconsis- share a common evolutionary ori- homology — or more tent? What if different gin. Even in a junior high school specifically, orthology GEORGE BELL scoring matrices produce biology class, one can very easily (separated by speciation) different results? We’ll define homology as features (such and paralogy (separated by gene probably try to optimize the details as genes, proteins, or even struc- duplication) — is a hypothesis that of our homology search for the spe- tures) that arise from the same reflects a history of shared origin cific use of these data, but our ancestral entity. Devising and that can be supported but not homology assignments will still be applying an operational definition unequivocally proven. We can open to debate. Also, unless this of homology that is practical and quantify similarity between pro- homology is well established, we’ll comprehensive, however, keeps a teins or gene sequences using per- want to make sure to explain our lot of biologists and bioinformati- cent identity, length of alignment, operational definition. cians quite busy. Here we discuss or even domain structure, but we some methods of assigning homol- can’t quantify homology; either Digging deeper ogy, along with some of our chal- features are homologous or they lenges that show why this is a aren’t. What if our operational definition problem that can’t be effectively Homology is commonly inter- of homology doesn’t turn up any addressed with basic sequence preted to mean present in the last orthologs of our favorite human comparisons. common ancestor, so even if all pro- gene using reciprocal Blast search? Homology has been described by teins evolved from the same good Has the missing gene just not been David Wake as “the central concept bits of primordial soup, knowing sequenced or annotated yet? Or is it for all of biology.” As bioinformatics this distant shared ancestry isn’t so actually missing from the genome

SEPTEMBER 2008 GENOME TECHNOLOGY 23

of our favorite model organism? tion, then there may not be any 1:1 genome environment is stronger How about if the human gene is orthologs. evidence, in addition to protein present by name in the other These 1:many or many:many and/or gene similarity, that genes are species? All of these possibilities homology relationships create really homologs and not just similar may need investigating. It would be extra challenges for comparing genes. On the other hand, alignment much easier for us if orthologs had genome-scale datasets across of genomes is not a solved problem, the same names in different species. On the other hand, if it and alignment gaps do not always species, but even some genes that appears that a gene duplication mean lack of homology. do have the same names don’t event occurred before speciation, The most powerful current meth- appear to be orthologous, now that we can try to resolve multiple ods use information from multiple we know more complete gene cat- homologs into 1:1 orthologs. All of species at once, and this orthology alogs. Some of these cases fit into this can be done better now than determination benefits from the the unfortunately-named category ever before, in part thanks to ever-growing number of genome of “functional homologs” which improved genome assemblies and assemblies. The Ensembl project, gene sets. This can reduce for example, leverages the power of The most powerful current strange observations, such comparative genomics by using as a recent look at a col- Blast to search with each gene methods use information laborator’s favorite gene in against all other genes (species by a fish. The fish genome species), clustering the similar from multiple species at assembly and gene predic- sequences, building multiple once, and this orthology tion pointed to this gene’s sequence alignments, and then presence in a set of a cou- generating phylogenetic trees determination benefits from ple dozen highly similar which can be compared to a species paralogs, a degree of gene tree. The use of sequences at differ- the ever-growing number of expansion that was absent ent phylogenetic distances helps genome assemblies. in other species. Further resolve a lot of cases that would be investigation led to the difficult to figure out with only a are proteins with similar functions much less interesting explanation pair of species at a fixed distance. but not of shared evolutionary ori- that the expansion of this repeat- This brings up the final way to gin (and therefore not actual flanked gene was very recent, hav- determine homology: consult a homologs). ing just occurred in the most reliable database that has already It would be very convenient for recent genome assembly. done the best possible large-scale biologists and database administra- Biomedical researchers who use homology analysis. These data- tors if all orthologs were clearly 1:1 mammalian model systems have a bases aren’t foolproof, but they’re a where, for example, one human much easier time identifying great place to start. gene is orthologous to one mouse homologs of human genes than Just as with other scientific state- gene. If analysis of the mouse others who experiment on worms, ments, we shouldn’t believe every- genome shows that virtually all flies, and yeast. First, the genes thing we read or hear about an human protein-coding genes have themselves have had much less time inferred homology relationship. If mouse orthologs, then why is it so to diverge, so the orthologs are they’re important to us, we probably hard to link every human gene to a much more similar. Second, the need to investigate the genes further mouse gene? Gene duplication genomes have had much less time to so we can hopefully convince our- and subsequent divergence, giving diverge, so chromosomes have selves and others: either they are rise to paralogs, can make determi- much longer conserved syntenic homologous or they aren’t. nation of orthology much trickier. blocks. As a result, if mystery gene If we discover two obvious human B is flanked by genes A and C in Fran Lewitter, PhD, is director of paralogs and two mouse paralogs, human, each of which have clear bioinformatics and research computing all of which appear to be orthologs A’ and C’ in mouse which at Whitehead Institute for Biomedical homologs, how can we figure out are close to each other on the same Research. This column was written in which mouse gene is the ortholog chromosome, we can look between collaboration with George Bell, PhD, a of each human gene? If gene these mouse genes to try to find B’. senior bioinformatics scientist in duplications occurred after specia- This conserved surrounding Fran’s group.

24 WWW.GENOME-TECHNOLOGY.COM______SEPTEMBER 2008

HIGH-PERFORMANCE COMPUTING Brute Force

Sweet Time for Informatics Following in the footsteps of genomics and proteomics, the budding field of glycomics is starting to show signs of getting its informatics act together. By Matthew Dublin

hile the nas- formatics core for the Consortium grated interfaces of diverse datasets cent field of for Functional Glycomics. “In a in the CFG’s relational databases, glycomics has cell, you have different glyco- which contain content on glycan- not received proteins, and each protein has mul- binding proteins, glycan structures, W nearly as tiple glycosylation sites. At each site, and glycosyltransferases. KEGG much attention and funding as its you have a variability of the type of Glycan is an extension of the Kyoto more established systems biology glycans that can be expressed, so Encyclopedia of Genes and Genomes siblings (namely, genomics and pro- you can see how complex it database and is managed and teomics), this small but steadily becomes when you want to know developed by the Kyoto University growing research community is every glycan at every glycosylation Bioinformatics Center. Glyco- solving its own database and soft- site of every glycoprotein in a par- sciences.de is maintained by the ware challenges. ticular cell.” German Cancer Research Center Just like genomics and pro- and provides researchers with mass teomics, glycomics has an “ome” as Glycan databases spec and glycan structure data as its holy grail — although this one well as applications for glycan is a long, long way off from com- Compared to both the availability analysis. pletion. The glycome is a complete and sophistication of databases and While certainly not impressive to map of all the complex carbohy- software tools for genomics and those familiar with current pro- drate or glycan structures in a par- proteomics, glycomics trails way teomics and genomics databases, ticular organism. These intricate behind. And just as each of those these repositories do mark an sugar structures have been shown has gone through its own database important development for gly- to play a key role in everything growing pains, glycomics must first comics. Prior to their arrival, the from pathogen recognition and get its data resources up to speed only major glycan structure sperm-egg interaction to immune before more researchers system response. In addition, and commercial vendors “If someone wants to many glycoproteins have been have a reason to start seri- identified as biomarkers for cancer ously contributing soft- decode the glycome of a and several diseases. ware tools to this area. Unlike DNA, RNA, or proteins, Currently, there are three cell, it’s a fairly compli- which are all template-driven, gly- major databases that house cated process. It’s not the cans are created by the actions of a glycan structure data: large number of enzymes, which KEGG Glycan, Glyco- same as someone saying, can result in a seemingly endless sciences.de, and a relational number of structural variations. “If database hosted by the ‘I want to decode the someone wants to decode the gly- Consortium for Functional genome or the proteome.’” come of a cell, it’s a fairly compli- Glycomics, an interna- cated process. It’s not the same as tional initiative funded by the resource available to researchers someone saying, ‘I want to decode National Institute of General Med- was CarbBank, a database hosted by the genome or proteome,’” says ical Sciences. The CFG’s database is the University of Georgia that Rahul Raman, director of the bioin- a Web portal connected to the inte- served as the de facto central repos-

SEPTEMBER 2008 GENOME TECHNOLOGY 25

itory for all glycan structures. bunny ears learning about HDTV. “I to a centralized and curated glycan CarbBank had its heyday in the think it’s fantastic that the format structure database. 1990s and has since run out of was agreed on, and that’s really “There’s really a need to get the sci- funding, although it provided a going to help,” says David Goldberg, entific community and the journals large part of the glycan structure a research fellow at the Palo Alto to agree on certain guidelines, so data and system architecture for two Research Center. “But it’s not used whenever someone wants to of the three newer databases. much — not because there’s any- deposit a structure they could just Though the three current data- thing wrong with the standard do it through a central submission bases share the same initial collec- itself, it’s just that there is not that system — and then that structure tion of glycan structure, they use much software out there yet that’s will automatically go to the different designed to take advan- large initiative databases,” says tage of it.” Raman. “All of us right now are try- “A lot of [glycan] structures Encouraging gly- ing to manually collect this informa- comics researchers to tion because there is no system to are still not represented adopt a standard file deposit a structure that will auto- among all these databases, format for glycan struc- matically be piped into the different ture data submission databases. That’s the main challenge and it will take time and would be beneficial not in maintaining and expanding the only to facilitate inde- current glyco-databases.” money for a repository like pendent database inte- GenBank for glycobiology.” gration, but also to Early tools make incorporating experimental data pub- Still, serious gains have been different file formats, a huge infor- lished in journals easier. “Each made since the formation of the matics stumbling block. KEGG individual database has made [its] CFG and other large-scale initiatives Glycan uses its own KEGG Chemi- own attempts to update their data geared toward mobilizing the gly- cal Function Format; Glyco- according to the literature, but it’s comics community. But Goldberg sciences.de uses the LINUCS for- hard because of the variety of nota- says that it’s a bit difficult to predict mat; and CFG uses a format tions used to represent glycan how long it will take for glycomics established by the International structures,” says Kiyoko Aoki- to catch up to proteomics and Union of Pure and Applied Chem- Kinoshita, an associate professor of genomics in terms of software istry. bioinformatics at Soka University in development. Many in the field feel Tokyo. “In general, it can be that this is due partially to the fact Setting standards assumed that a lot of [glycan] that glycomics is still too small a structures are still not represented sector of the market for commercial In September 2006, a workshop among all these databases, and it developers to care about, although was held at the National Institute of will take time and money for a a handful of vendors have started Health so that glycobiologists from repository like GenBank for glyco- offering some tools. Proteome Sys- across the globe could assess bioin- biology to be developed.” tems has a glyco-database and a formatics needs and the current Aoki-Kinoshita and others believe suite of software tools for analyzing state of glycan structure analysis that the most urgently needed mass spec data and structure pre- tools. An outgrowth of this meeting improvement is the consolidation of diction. And Premier Biosoft Inter- was the establishment of a standard these databases along with supple- national is also pitching SimGlycan, file format for exchanging glycan mentary data such as pathways, its mass spec software analysis tool structure data. They chose the interacting proteins, and binding geared toward studying glycosyla- GLYDE-II XML file format, devel- affinity into a one-stop resource. tion, a key area of post-translational oped by William York, an assistant The creation of such a resource was study for glycobiology that looks at professor at the Complex Carbohy- also deemed a priority at the 2006 when glycans attach themselves to drate Research Center at the Univer- NIH meeting, as leaders in the field proteins. Still, many glycobiolo- sity of Georgia. And while this is hope that a standardized glycan gists agree that, for now, most of the certainly exciting to many, it’s like a structure data file format such as software development will come bunch of TV owners still using GLYDE-II XML will eventually lead from academia.

26 WWW.GENOME-TECHNOLOGY.COM______SEPTEMBER 2008

Along these lines, Goldberg has ing. GlycoVault is hosted by the researchers like Goldberg and oth- developed an automatic annotation University of Georgia’s Integrated ers with the onus of providing software tool called Cartoonist that Technology Resource for Biomedical tools to the small but growing works with single MS data to deter- Glycomics, an initiative funded by community. mine the composition of a particu- the National Center for Research And as was the case in the early lar glycan structure. The program Resources. This application also days of genomics, once that divide works by selecting the most plausi- contains the Glycomics Browser, a between computer scientists and ble annotations for each peak in a Web-based visualization and analy- bench biologists is traversed and the mass spectra profile from a library sis tool for glycan data. databases become more developed, of possible cartoons. Goldberg says Overall, the lack of software and things will ramp up. “I think that the current version of Cartoonist is the glyco-informatics community’s once the data can be accumulated, unique among software tools; ear- small size may be a sort of vicious the bioinformatics fields can start to lier research in glycomics utilized cycle hampering its growth. “It’s a develop methods specifically for MS/MS because researchers were chicken or egg problem. Because glycobiology, but it is important for merely copying the same tech- the [glycomics] community is informaticians to work closely with niques that worked in proteomics. small … there isn’t much of a experimentalists in order to “From single MS data, Cartoonist demand for software,” Goldberg develop useful tools,” says Aoki- lets you figure out what the glycans’ says. “But on the other hand, if Kinoshita. “The language barrier compositions are, and then it there was better software, maybe between bionformatics and glycobi- makes a very good first guess at more people would do this kind of ology needs to be broken down what the actual structures [are],” experiment, so I think it’s going to [and] it is my hope that this can be says Goldberg. At the moment, ratchet up slowly.” This leaves overcome in the near future.” those wishing to use Car- toonist must send their spectra directly to Gold- berg, but he says that will DISCOVERING change in the next year as he * works out the kinks and *BIOMARKERS * beefs it up to include IS AS EASY MS/MS data. He hopes to * distribute the tool to CFG AS IDQ. members, and then ultimately to make it more widely distributed. “The * tools used to assist in the * annotation of glycan mass spectra have made major contributions to this field,” says Aoki-Kinoshita. “In particular, the Cartoonist suite of software, which is TargetIDQTM Services being used by the CFG to Deﬁning the future of Metabolomics annotate the large amount of

data they are generating, Biocrates’ services enable you to identify and quantify has been apparently very metabolomics biomarkers in a broad range of ﬂuids useful.” and tissues. Our assays are robust, reproducible, Over at the CCRC, Glyco- and use the least amount of sample in the industry. CREATING KNOWLEDGE Vault, a Web-based infor- Metabolomics Society, Boston, MA FOR HEALTH matics gateway that con- September 3-6, 2008 tains databases, ontologies, Optimizing Cell Culture Development, Boston, MA [email protected] and other glycan structure- September 15-16, 2008 www.biocrates.com related data, is also promis-

SEPTEMBER 2008 GENOME TECHNOLOGY 27

Under One Roof BASIC MEETS CLINICAL

Starting from Scratch Ken Stuart founded the Seattle Biomedical Research Institute with a single grant and high hopes. Thirty years later, a 260-member staff and a $40 million budget suggest he did something right. By Meredith Salisbury

et’s say you won a grant worth $140,000. What would you do with it? LChances are, parlay- ing that funding into your own fledgling research institute would be pretty far from your mind. But that’s just what Ken Stuart did in 1976, when he was in his mid- 30s and received his first NIH grant. It was a modest, two-year award at $37,000 per year (adjusted for inflation, that’s worth about $140,000 today). He used the money to rent space in an office park, start up his research lab focusing on global infectious disease, and pay his salary. It was a humble beginning for the Seattle Biomedical Research Institute. SEATTLE BIOMEDICAL RESEARCH INSTITUTE Stuart says that his early training experiences had provided exam- were attracted by the environment make the transition to “a full- ples, both good and bad, of differ- that I’d created,” he says, and over fledged research institute with an ent types of research environments. the next decade the institute came integrated vision and mission.” A stint at the National Institute for to be known as a place to do inten- They made the transition. Medical Research just outside Lon- sive science. But with that empha- In early 2004, SBRI moved into a don showed him an institution that sis on science came a lack of over- new $40 million facility, built after “enhanced the ability of scientists to all goals for the institute as a whole. a major fundraising effort. Today, conduct research,” while a teaching “The way the institute developed, it the institute occupies some of the gig at a Florida university made really was sort of a collection of space and leases out the rest to clear the distracting duties of independent laboratories,” he says. researchers from a local children’s teaching and other administrative By the ’90s, Stuart had become hospital as well as some companies. necessities. His vision was simple: interested in pursuing infectious Stuart says that he expects the to create a “research environment disease research beyond the basic institute, which has been growing at that was efficient and effective” — a lab and wanted to focus on transla- about 25 percent annually for the place where research was first and tional work. Internal discussions past five years, to eventually take foremost, and any other tasks were over time revolved around whether over the entire building. Today, kept to a minimum. to remain as a series of “boutique SBRI boasts a staff of 260 with 15 Stuart’s gamble paid off: “People laboratories,” says Stuart, or to principal investigators; Stuart plans

28______WWW.GENOME-TECHNOLOGY.COM SEPTEMBER 2008

to increase that over time to 400 for seven days before spreading out full-time employees. >SEATTLE BIOMEDICAL into the bloodstream and causing RESEARCH INSTITUTE infection. Using functional genomic Need for translation Seattle, Wash. studies and mouse models, Kappe and his team were able to determine While getting scientists to buy DIRECTOR: Ken Stuart the gene expression pattern during into the vision of one institute ESTABLISHED: 1976 this stage for the first time. Knock- instead of many labs might have SIZE: In March 2004, the SBRI team out studies proved that deleting cer- taken some discussions, Stuart moved into a new $40 million, five- tain genes at this stage left the para- says the real challenge in the story facility. The institute currently site unable to complete its life cycle course of the group’s development leases out space to researchers from and infect the host. In mouse mod- has been getting people to embrace Children’s Hospital & Regional els, dosing the mice with this genet- translational research. At the very Medical Center and to companies, ically engineered version of malaria beginning of the institute, this but planned expansions to the staff induced a “very powerful protective wasn’t even an option, he says. In will likely have the institute take over immune response” — such that the mid-’70s, studies on infectious the whole building in the future. when the mice were then infected disease were so early-stage that “I STAFF: During the past five years, with full-strength malaria, the mice could not really see a way to do SBRI has been growing at about 25 proved to be completely immune research that would support the percent annually, bringing its head- over the course of their lifetime. Dur- activities that would lead to inter- count to 260 employees (not all of ing the upcoming clinical trials, the ventions,” Stuart recalls. whom are full-time). The institute same process will be performed on But in the past decade or so, that plans to increase that to 400 full-time human patients to see if the immune has changed radically, especially in employees. There are 15 PIs on staff. response holds true. the areas that SBRI scientists focus FUNDING: The institute’s current Another potential malaria vaccine on: HIV, tuberculosis, malaria, and annual budget is $40 million, almost comes from Patrick Duffy’s group, emerging infectious disease. The all of which comes from grant fund- which is studying pregnancy real problem was that the public ing, though the SBRI team has begun malaria. The team has identified a funding system “didn’t reward” to focus more on fundraising with key protein used by parasites in the translational work, Stuart says; sci- philanthropic organizations. placenta, and are aiming to develop entists are rewarded for basic FOCUS: The main program areas a vaccine to target the protein. research and for publishing. “We are HIV, malaria, tuberculosis, and had many discussions about emerging infectious disease. Going global [working toward an intervention] CORE LABS: SBRI’s cores include — why that’s strategically impor- technologies for DNA sequencing, As he was getting the institute tant, why it’s morally important.” imaging, protein production, pro- going, Stuart realized that his Today, SBRI scientists are months teomics, bioinformatics, and more. group would never be large away from clinical trials for a COLLABORATORS: SBRI places a enough to have expertise in all areas malaria vaccine, and they have strong emphasis on partnerships with of infectious disease — so collabo- other promising candidates for other institutions to help extend its rations have been essential to HIV and malaria in the pipeline. reach and capabilities. The group has SBRI’s development. Currently, for The first one, which is expected to more than 100 alliances, with part- instance, many of the partnerships enter trials in 12 to 18 months, ners including Microsoft, Harvard, help the institute connect with began with work by malaria expert Walter Reed Army Institute of people who are experts in develop- Stefan Kappe, who joined the Research, the Pasteur Institute, and ing vaccines. The institute’s website institute five years ago. He says a Novartis, to name a few. lists 125 collaborations with part- significant lure of SBRI was that as ners ranging from New York Uni- an independent institution — versity to Novartis to the University rather than, say, a program within vaccine was part of the goal of the of Nairobi. “From our total budget,” a medical school — it’s “very nim- malaria program, so he focused his Stuart says — this year, that’s $40 ble” and “we can shift our priorities research on the liver stage of the par- million — “about half of it goes out very rapidly.” asite, which is when it enters its the door to support our collabora- Kappe knew that working toward a human host but remains undetected tive activities.”

SEPTEMBER 2008 GENOME TECHNOLOGY 29

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METABOLOMICS

niques and technologies and protocols, and some it is reproducible Taming and some of it isn’t. It’s a bit of a Wild West, but there’s a bit of consolidation and the trends are there,” Metabolites says the University of Alberta’s David Wishart, who heads up the Human Metabolome Project. “I Metabolomics studies inch scientists ever closer think in a year or two it will be a more mature field with more con- to understanding phenotype. But to really make solidation and more consistency and more robustness.” progress, pioneers are working on improving the Detection technology and analytical tools of the field. In metabolomics, the divide has been between nuclear magnetic resonance and mass spectrometry. Both tools can identify a sample’s BY CIARA CURTIN metabolite population to varying degrees of success. More and more, 1966 Biochemical though, researchers are combining Journal article the approaches to take advantage of recently unearthed their strengths while minimizing by Gary Siuzdak’s their weaknesses. At the same time, Alab at the Scripps people are using new tools and Institute looks to be the first methods for both separation and metabolomics experiment — detection to take a gander at their though they certainly didn’t call it metabolome of choice. that then. In it, researchers from NMR, which dates back to the Baylor College of Medicine describe 1940s, has long been used by using gas-liquid chromatography to chemists to identify molecules in a separate metabolites from urine and sample. As a tool, it gets kudos for tissue extracts. They also add that GARY SIUZDAK being stable, reliable, and robust. GC-coupled mass spectrometry “In NMR, you can analyze the same gives a “diagnostic tool of great to data analysis to metabolite iden- sample today and this time next power” — something today’s tification, metabolomics as a field is year and get a very similar result,” researcher already knows. But also still trying to resolve robustness, says Warwick Dunn at the Univer- in that article, C.E. Dalgliesh et al. identification, and analysis issues sity of Manchester. grumble about overlapping peaks, that the other disciplines within sys- The tool has a variety of roles in resolving those peaks, and the lack tems biology have already over- the lab. For one, it can be used to of a database housing known come. Metabolomics, though, is get a first look at what the metabolites. Sound familiar? working hard to live up to its poten- metabolome contains. “NMR is our Today’s metabolomics researchers tial and join the ranks of the high- main technique, which we would have the same gripes, but are better throughput fields. Researchers are use first as sort of a survey tech- poised to do something about capitalizing on the track record of nique,” says John Lindon, a profes- them. The technology isn’t very fast NMR and the sensitivity of mass sor at Imperial College London. or robust as compared to other big spectrometry to increase the number Or it can be used for metabolite players in systems biology, and of metabolites detected and then profiling. “NMR is very good identifying all the metabolites in a identified through new databases. because it tells you exactly which sample can be nearly impossible. At “When it comes to metabolomics, position in a molecule contains a 13C each step, from sample preparation it’s just a plethora of different tech- or 15N, whereas mass spec only tells

SEPTEMBER 2008 GENOME TECHNOLOGY 31

Things to consider before purchasing a Next Gen System. you how many positions are labeled, metabolomic technologies. “The mass not which ones,” says Andrew Lane, a spec technology is wonderful now. professor at the University of The robustness is great, especially the Louisville’s James Graham Brown Can- new time-of-flight and quadrupole SCALABILITY cer Center. time-of-flight mass spectrometers. Throughput advances without And, of course, NMR has its down- They have improved dramatically in costly upgrades side. “The big knock about it is that it’s the last couple of years,” says Siuzdak. not very sensitive,” says Wishart. Not only is the detection step being Indeed, according to Wishart and improved, but advances are also com- THROUGHPUT Siuzdak, an NMR-based characteriza- ing along on the separation side. Ultra More than 6 GB and 240M tags per tion of a tissue sample or biofluid high-performance liquid chromatogra- run for cost-effective, large-scale yields a little more than 50 molecules, phy came on the scene a few years ago, resequencing and tag applications but looking at that same sample with using higher pressures and smaller par- mass spec methods can yield hundreds ticle sizes to increase resolution and or even thousands of molecules, sensitivity, allowing scientists to detect FLEXIBILITY depending on the chromatography even more metabolites. “The more Two independent ﬂow cells to run technique coupled to the mass spec. things you can see, the greater multiple applications in a single or Some scientists use NMR for surveying, overview you can get of the biology of staggered run and then apply mass spec for a more the system,” Dunn says. He’s not the targeted analysis. only one looking into UPLC: Lindon Manchester’s Dunn focuses on mass and his group have begun to couple it ACCURACY spec — particularly liquid-chromatog- with time-of-flight mass spectrometry. System accuracy of greater than raphy mass spec — and he works on Another separation approach that is 99.94% for the power to detect developing and optimizing methods to catching on is HILIC. Hydrophilic rare, causative SNPs without high use it in metabolomics. While mass interaction chromatography allows false positive rates spec may be able to see more metabo- researchers to detect more of the lites than NMR, it has its own draw- small, hydrophilic molecules that backs, primarily reproducibility. In his often are removed during a wash or MATE PAIRS lab, Dunn says, two separate sample that come out at the very beginning of Libraries with inserts ranging from sets might give 50 interesting metabo- the separation. Siuzdak is particularly 600 to 10,000 bp for resolution of lites, but only 10 of them overlap. intrigued by this method. “I’ve been structural variation “In an ideal world, you’d use both recently surprised by some of the technologies because, in any analytical results that we’ve been getting that’s technology, there is some bias in what allowed us to certainly see new DATA SETS it can detect, whether it be the type of things,” he says. He is currently using Publicly available human datasets for metabolite it can detect or the sensitiv- HILIC to try to detect new molecules in accelerating biological discovery and ity, for example,” Dunn says. knock-outs. “It’s just another window In particular, he works on increasing into these samples,” Siuzdak says. analysis tool development the reproducibility of mass spec by using an automated closed-loop strat- Deconvolution SUPPORT egy that has minimal human intervention. Over many iterations, the Robot The data that comes out of the end of Trusted service and support team Chromatographer, as his team calls it, NMR or mass spec is a mess of peaks available when and where you initializes the instrument settings and and spectra. Making sense of all that need them then changes them as it cycles through can require some serious analysis, looking for the optimal settings. though some old hands can recognize When used on GC-TOF mass spec, NMR peaks just by looking at them. Dunn and his colleagues increased the Most scientists rely on software pack- number of peaks seen by three-fold. ages to resolve the curves and decon- Newer technologies are also coming volute the data into something resem- onto the scene to topple NMR, LC/MS, bling a list of metabolites. “If you use and GC/MS from the top spots in chromatography on very complex

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samples like urine or plasma or analyze mass spectrometry data for metabolites, you still won’t be able something, there would be so many metabolite profiling. Their XCMS is to identify all the compounds, or all small molecules and metabolites an open-source data analysis soft- the peaks,” Wishart says. eluting at pretty much similar ware package for LC/MS data that A few database projects — chromatographic times that you get not only peak-picks, according to including efforts by Wishart and overlapped peaks, which makes Siuzdak, but uses endogenous Siuzdak — are attempting to index them difficult to quantify and it metabolites found in all the and curate all the known metabo- makes it difficult to identify what it datasets as internal standards and lites. “Unlike in proteomics or was,” says Henrik Antti, an associ- aligns the peaks based on ate professor at the University of the retention time. Umeå in Sweden. Different research Then, XCMS looks “We can use that statistical groups are developing new and bet- through its analysis and ter software to help deconvolute find the peaks that correlation to prove those what’s in a metabolome. change between the two peaks are linked across At Imperial College, Lindon and dataset that are statisti- his colleagues have developed and cally relevant. “So now hundreds or even thousands are using a statistical analysis you have a set of mole- method to identify NMR peaks. “It’s cules, typically, that look of samples.” not like a gene chip where you have very interesting,” says one spot equals one gene,” he says. Siuzdak. He and his colleagues genomics where we can say we “Here, a molecule, a metabolite will also recently came out with XCMS2 know all the amino acids and all the give many peaks on the NMR spec- for MS/MS data. bases and therefore the library or trum. We can use what we know For researchers blending NMR the alphabet is known, the alphabet about NMR to identify where those and mass spec data, Lindon and his isn’t really fully known for all the come from.” colleagues have also been working things that we expose ourselves to,” Building on a previous tool, called on a tool that bridges the NMR- Wishart adds. TOCSY (for total correlation spec- mass spectrometry divide. Their Starting in January of 2005, troscopy), Lindon’s team made a statistical heterospectroscopy, or Genome Canada funded the tool called STOCSY. This new SHY, works to put NMR and Human Metabolome Project; part of method takes advantage of the cor- UPLC/MS data from the same sam- its mandate was to catalogue and relation between peaks in NMR ples together by analyzing signal consolidate all naturally occurring spectra — that multiple peaks can intensities from the molecules as metabolites. It contains about 2,500 come from the same molecules and detected by the different methods. metabolites, culled from the litera- always occur in proportion. As an “You get a bit of information from ture and confirmed with NMR, example, Lindon points to lactate, the mass spec and a bit of informa- LC/MS, or GC/MS, as well as from which has two NMR peaks — one tion from the NMR, you can put the the group’s own experimental data. from the methyl group and one two together to identify molecules,” Siuzdak and his colleagues are from the CH group. Since NMR says Lindon. working on Metlin, a depository detects the hydrogen atoms of these for mass spectral metabolite data. groups, these two peaks will always Databases It currently contains about 23,000 be in a proportion of three to one. molecules, and Siuzdak says they “We can use that statistical correla- With the molecules in hand, the are adding more to it constantly. tion to prove those two peaks are identity of the metabolites can The 1966 paper, says Siuzdak, linked across hundreds or even begin to be uncovered, though it said the main problem with using thousands of samples,” Lindon isn’t always possible when they GC/MS was that there are so many says. This relationship can help don’t correspond to a known molecules that are unknown and researchers work out which peaks metabolite. “There are still a lot of there’s no comprehensive data- of an NMR spectra go with which unknowns in terms of compounds base. “What happens since then is and, Lindon adds, help them iden- that people see or identify. If you now there’s a database that has well tify potential biomarkers. were to take a sample from a person over 10,000 molecules in it,” Siuz- At Scripps, Siuzdak and his col- or a plant and use our standard dak says. leagues developed their own tool to libraries of known endogenous While these projects and others,

34 WWW.GENOME-TECHNOLOGY.COM______SEPTEMBER 2008

such as Riken’s SpinAssign and the But that integration across the sense of data that we collect at the Madison Metabolomics Consor- field is a challenge, not only for different levels of the ’omics — tium Database, have made progress metabolomics, but for systems genomics, transcriptomics, pro- in cataloguing metabolites, esti- biology as a whole. “There’s teomics, and metabonomics — mates place the number of metabo- absolutely no point in just concen- understanding all of that in the lites in the tens of thousands. The trating on one ’omics. We have to be context of systems biology is very, databases have a long way to go able to integrate data across all the very important. It’s where we’re before they can be considered any- ’omicses,” Lindon says. “Making going.” thing close to exhaustive. “[Metabolomic databases] still have a ways to go. They are not as robust as Blast or Mas- cot,” Wishart says. Not alone

Metabolomics isn’t the be- all and end-all. Once the data is gathered and ana- lyzed, with the metabolites identified, metabolomics often leads to new questions that can be followed up by using the other arms of systems biology. Because metabolomics may be more reflec- tive of phenotype, as Siuzdak says, using it in combination with “the genetic information that we have, it gives us a really interesting story.” Andrew Lane agrees. “You can’t do just one of the ’omics on its own,” he says. “Once you’ve found something out from a metabolic pathway, you need to go back and ver- ify that, OK, we’re positing that this metabolic pathway has increased activity, that implies that there’s either increased gene expression for those enzymes in that pathway or that some of the HD qc tools enzymes in that pathway ready integrated have become more active by post-translational modifica- tion or by allosteric regulation. You have to look at gene ______expression, protein level, and protein post-translational modifications.”

SEPTEMBER 2008 GENOME TECHNOLOGY 35

Connect

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STRUCTURAL VARIATION

Lipmann Institute focuses on the 350 KB region at 8p23.1 in the Counting on human genome, a cluster of well- researched defensin genes that have extraordinary range in copy Copy Number number variation. “The range of variation is really huge, from two copies as a normal diploid genome Research into copy number variation is highlighting to up to 12 to 14 copies of that entire region,” he says. The question just how complex genomic differences are. is, how can people vary by so many bases of sequence and still show no A glimpse at the latest findings and approaches to difference in phenotype? An answer may lie in understand- understanding CNV regions and what they mean. ing how copy number variation happens, but that’s elusive at this point, says Guryev. “Finding the molecular mechanisms responsible for CNV formation remains [a] chal- BY MEREDITH SALISBURY lenge. Change in copy number is caused in various ways, such as non- opy number varia- allelic homologous recombination, tion broke onto the non-homologous end joining, insta- scene four years ago, bility of tandem repeats, or transpo- when what was once sition of mobile elements,” he notes. Cthought to be an “We still do not have a complete occasional genomic glitch turned overview on how these mechanisms out to be an incredibly common contribute to the diversity of struc- mechanism of DNA variation. tural genome alterations.” Charles Lee, an associate professor Some light has been shed on the in the pathology department at issue by research such as Noah Harvard Medical School, worked on Rosenberg’s, which focuses on CNV one of the earliest studies revealing in human populations. Rosenberg, this phenomenon. A research proj- CHARLES LEE an assistant professor at the Univer- ect involving 39 healthy control sity of Michigan, has been studying patients showed a significant num- Today, scientists posit that variation populations globally to determine ber of “gains and losses which we in copy number has even more of a patterns of variation. “For the most weren’t expecting to find,” Lee says. role in disease association than part, the pattern of copy number “We thought it was artifacts, [but] SNPs do. variation in worldwide populations when we started validating them, matches what we expect in terms of they weren’t artifacts. They were Basic biology SNPs and microsatellites,” he says. true gains and losses.” “That’s telling us the history of copy The real surprise was how much of Much of the research into CNVs number variants largely matches an impact these variant regions were in these early days is simply the human history as a whole.” having. “What we did not anticipate geared toward understanding Rosenberg’s work also demon- is that copy number variations are so what these elements are doing, strated evidence of natural selection abundant that they affect [a] greater how they came to be, and how it’s at work on these variants. “We number of bases than single possible for organisms to have noticed that many of the copy num- nucleotide polymorphisms do,” says such different copy numbers and ber variants were rare,” he says, indi- Victor Guryev, a member of Edwin still appear the same. cating that “there’s some negative Cuppen’s lab at the Netherlands Matthias Platzer at the Leibniz selection operating against at least a Institute for Developmental Biology. Institute for Age Research and Fritz reasonable fraction of these variants.

SEPTEMBER 2008 GENOME TECHNOLOGY 37

Otherwise, some of these would be ments done on these organisms five, and you have no information a bit more common.” and will have to be controlled for, about the exact copy number,” he Another piece of the puzzle was he adds. adds. His team worked with MRC supplied by Harvard’s Lee, whose Edwin Cuppen’s group is using Holland to develop MLPA, or involvement with the Structural inbred rat strains to try to get a purer multiplex ligation-dependent Genomic Variation Consortium — a view of copy number variation. probe amplification, a technology partnership with Harvard, Sanger, Evaluations of these regions in rat specifically designed for the and Toronto’s Hospital for Sick are still at a low-resolution phase, defensin gene region he studies. Children — led to research into Cuppen says, but he believes the MLPA increases probe density to get population differences of copy method of using inbred strains will a high-res view of the region, and number in amylase genes, which are remove a lot of the background Platzer says that “from our point of involved in starch digestion. As noise that can’t be controlled in most view, this is the most quantitative hypothesized, populations with organisms. So far, he says, it’s clear approach at the moment.” higher levels of starch in their diets that copy number changes are Rosenberg says there’s still a need had higher numbers of the gene. responsible for “quite a few expres- for better quality control, espe- “That was the first time one of these sion differences” in the organism. cially to reduce false positives, and CNV regions was shown to be that technology needs to evolve to under positive selection,” Lee says. Complex techs account for more complexity. His population study looked for five Move to models Cuppen notes that copy number states of variation — homozygous events are more complex than ini- or heterozygous deletion, normal, Of course, biologists are taking tially suspected. Copies don’t and homozygous or heterozygous advantage of their go-to resource for appear faithfully and in whole; addition — and he says current better understanding bizarre events they can be duplications combined tools make it difficult to go beyond in the human genome: CNV with inversions and small dele- those states. research into model organisms is tions, for instance, making them taking off. Lee says that the turn to much more difficult to detect com- Link to disease animal models lagged; when he prehensively. Because of that, Cup- began to study variation in chimps pen uses a number of technologies Whether technologies improve or and macaques, “I got a sense … that to study these elements, and says sequencing gets cheap enough to there was clearly a deficiency of that just one platform isn’t enough enable whole-genome scans for copy work being done in other animals,” to track this kind of variation. His number variation, the ultimate aim is he says. A recent paper from his group uses standard array CGH the same: figuring out how these team showed a first-pass look at technology with paired-end changes contribute to disease. these primates, demonstrating that sequencing as well as optical map- Research has already shown that copy number variation does indeed ping for the rat studies. CNVs are tied to schizophrenia and affect their genomes. As it turns out, “A promising new technology for autism, among other diseases, and Lee says, “when you have segmen- detecting structural variants is com- scientists expect that trend to pick up tal duplication in the genomes of bination of paired-end mapping steam. This “highlights the impor- organisms, they do have the ability and next-gen sequencing,” Guryev tance of copy number changes in dis- to foster the creation of copy num- says. “However, it will require even ease etiology,” Guryev says. ber variants.” higher sequencing throughput and In this sense, CNVs are “like risk Following that, Lee’s group has price reduction before we can use it factors,” says Lee. They may eventu- been delving into zebrafish, which for such applications as diagnostics ally help stratify patients to show also has segmental duplication. His or association studies.” which will respond better to one results aren’t yet published, but Lee One challenge is that standard drug than another, for instance. believes the level of variation he’s technology — generally speaking, Lee believes that a connection to seen in zebrafish will be “a very eye- array CGH — isn’t precise enough cancer is imminent. “I think we’re opening experience” for the to quantify copy number variation, going to find that there are some research community. So much vari- says Liebniz’s Platzer. “In these CNVs” that increase predisposition ation could play a significant role in techniques you see just that there to cancer, he says. “I think it’s com- the run-of-the-mill genetic experi- are more than two, or maybe four or ing right around the corner.”

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ALTERNATIVE SPLICING A Complexity that Goes Beyond Genes

Most genes are subject to alternative splicing, but it’s still early days in understanding the phenomenon across pathways or on a genome-wide scale. A look at some pioneers in the field and the technologies they’ve commandeered to make sense of it.

BY JEANENE SWANSON

DIANE LIPSCOMBE AND MICHAEL WOLFE

40______WWW.GENOME-TECHNOLOGY.COM SEPTEMBER 2008 PHOTO: SAM RILEY

or most of the past large-scale tools like splice arrays 10 years, Michael and up-and-coming tools such as Wolfe has been multiplexed PCR and high- studying the bio- throughput sequencing are just Fchemistry of beginning to enter the alternative gamma-secretase, painstakingly splicing research world. detailing its structure, function, and mechanism of action. The Protein by protein main culprits of Alzheimer’s disease are short amyloid-β peptides that Like Wolfe, Brown University’s build up and clump together in the Diane Lipscombe uses splicing as a brain. Gamma-secretase works window into the expression diver- alongside beta-secretase to cleave sity of her protein of interest, the amyloid precursor protein into voltage-gated calcium channels. its fatal, truncated cousin. Several “It’s very anecdotal. Her lab was one of the first to clone years ago, Wolfe began characteriz- these proteins in neurons, where ing beta-secretase, but this time People are working on they’re important in modulating with a new tack: determining how their gene of interest, processes as diverse as gene tran- alternative RNA splicing affects the scription and neurotransmitter enzyme’s function. Recently, Wolfe and they find different release, and have been linked to published work that identified epilepsy and migraines. In her cur- alternative splicing events in beta- isoforms by cloning the rent studies, Lipscombe looks at secretase. genes and [then] try to how neuronal-specific factors affect “It’s starting to look like most splice variation in the channel and genes in the human genome are figure out what this end up fine-tuning its structure and alternatively spliced,” says Wolfe. A behavior. She typically uses gene single gene can generate many other isoform is doing.” databases and comparative types of proteins, which lends the Benoit Chabot sequence analysis to identify a hit, relatively small human genome its UNIVERSITY OF SHERBROOKE and then goes back in with PCR to extreme diversity of expression. see how the splice isoform may be That beta-secretase undergoes While there’s a lot of interesting differentially expressed in the tissue splicing is not surprising. “It seems pathway analysis being done, the of interest. “It’s very old-fashioned,” to be a key regulatory event, and a tools to look at this kind of variation she says. way for the cell to control what are, for the most part, in early In work published in 2004, Lip- kinds of proteins are produced stages. Most researchers still use RT- scombe found that a particular iso- from a single gene,” Wolfe says. PCR to identify new splice variants form of the channel was enriched in Alternative splicing has now been or to confirm splice microarray nociceptors, neurons that can sense found to exist for at least 70 percent results. Biologists have only just and signal pain. She noted in a of genes. After DNA is transcribed begun to scratch the surface of recent study that this channel is not into an mRNA precursor called associating different isoforms with only more sensitive to neurotrans- pre-mRNA, splicing machinery unique functions inside the cell. mitters, but it’s also more sensitive known as the spliceosome steps in. “Looking at diversity of function is to opiates like morphine. “We’ve The spliceosome snips out the still in its infancy. It’s very anec- long thought that the expression of introns and patches up the strand to dotal,” says Benoit Chabot at Que- different splice isoforms probably form a finished mRNA, which is bec’s University of Sherbrooke. underlies a lot of the differential then translated into a protein. In “People are working on their gene of effects of drugs in different path- alternative RNA splicing events, interest, and they find different iso- ways,” she says. certain exons are skipped, or spe- forms by cloning the genes and Alternative RNA splicing can vary cific introns left in, so that the final [then] try to figure out what this depending on tissue and stage of mRNA sequence can vary, produc- other isoform is doing. Nobody is development, among other things, ing different splice isoforms of the doing it in a systematic manner and in most circumstances there is same protein. right now.” For the most part, a healthy balance between differen-

SEPTEMBER 2008 GENOME TECHNOLOGY 41

tially expressed isoforms. Existing be specific enough to distinguish side by side in certain percentages, between the tumor suppressor and variant transcripts can regulate its oncogenic variant. gene expression by turning protein Brown’s Lipscombe, however, says manufacture on or off. When aber- the hardest part isn’t identifying the rant splicing occurs — for variant at the RNA level. “Where we instance, in some cancers — path- are incredibly limited, where we ways commit signaling errors have a huge hurdle to overcome, is which lead to downstream trouble. to be able to distinguish at a protein Biologists are looking for not just level,” she says. “It’s a problem. You whether a particular isoform is don’t have that much control.” As an there, but whether it’s an actual example, there is a calcium channel splice variant or just an error in variant differing by an exon that transcription, whether it’s tissue- encodes only two amino acids, but specific, and whether it occurs at a SCOTT FRIEDMAN Lipscombe hasn’t yet been able to level that’s meaningful enough to create antibodies for the two nearly have any noticeable effect. What they found is SV1, “which is identical isoforms. As a stopgap really composed of three of the four solution — it’s harder, it’s more Peering into pathways exons of the tumor suppressor, expensive, but it gets the job done seems to be drastically up-regulated — she’s created mice that express Scott Friedman, chief of the liver in many late-stage cancers,” says co- one or the other. disease division at Mount Sinai author and fellow Mount Sinai sci- Despite how difficult it may be, School of Medicine, knows about entist John Martignetti. He adds picking up the subtle differences devoting a lot of time to a single that there seems to be an interaction between isoforms is one of the gene. For more than a decade, he’s between the two variants depending most important steps in pathway been studying alternative splicing in on how much of each is present. analysis. “By having recognized the a tumor suppressor gene called Friedman and Martignetti recently importance of splicing in [the Krüppel-like factor 6 and how a collaborated on work that fleshes KLF6] gene, it greatly sensitized our splice isoform, KLF6 SV1, affects out some of SV1’s mechanism of interest in looking for splicing as an liver cancer. He cloned the gene for action; using RNAi, they were able explanation for other biologies, KLF6 10 years ago, and after his to show that knocking down the Ras using different genes,” Friedman research turned toward alternative signaling pathway could decrease says. “Once you recognize it’s splicing, found that SV1 antago- SV1 production and inhibit tumor another level of regulation and you growth. Martignetti’s work look for it, it’s amazing how preva- “Once you recognize it’s also looks at prostate and lent these kinds of regulatory path- ovarian tumors, and what’s ways are.” another level of regulation going on in signaling dys- regulation in these can- Microarrays to market and you look for it, it’s cers. He’s seen up-regula- amazing how prevalent tion of SV1 in prostate While tool development is still cancer, where it appears to underway for this field, genome- these kinds of regulatory play a role in many differ- wide analysis is definitely in swing. ent pathways. “It seems to In fact, several vendors are hard at pathways are.” be involved in changes in work co-opting gene expression proliferation, in metastasis, arrays to study alternative splicing. nizes full-length KLF6 suppressor and even in angiogenesis,” he says. Affy’s Exon Array has become pop- activity. “Transient splicing can be a To detect and measure different ular for genome-wide expression very subtle fine-tuning mechanism splice variants, they also use PCR analysis, and while ExonHit Thera- for adjusting cell function,” he says, and are trying to develop splice- peutics and Jivan Biologics offer “whereas in cancer, it looks like it’s specific monoclonal antibodies. both exon and exon splice junction kind of a constitutively on switch RNAi using siRNA has been a very arrays, the latter have proven to be that affects cells.” effective tool, they say, in that it can the truly useful tool in the toolbox.

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Jivan’s Jonathan Bingham says that you don’t have as much flexibility to will be making refinements to its while arrays that probe exon bod- do that much coverage [with] RT- software in the future. Most of their ies give you a lot of information, PCR,” Jaskowiak says. initial customers have been aca- “you’re left with the problem, how To create its arrays, ExonHit demic researchers, but Bingham do those pieces fit together? And mined EST databases and other says he’s seen a shift toward using you get that answer more directly if cDNA genomic information, and the arrays for diagnostic purposes you have probes for the splice then partnered with Affy and Agi- like studying biomarker signatures, junctions.” lent to print them. They offer drug response, or toxicity. And Because most genes are alterna- genome-wide arrays for human and with the ability to look across many tively spliced, says ExonHit’s John mouse, as well as other arrays spe- genes at once with arrays, it’s easier Jaskowiak, “you need to start taking cific to druggable-target gene fami- to study splicing regulation — a a higher degree of interrogation of lies, such as apoptosis, cytokines, or separate field that looks at hundreds the genome.” ExonHit has been kinases. of interacting genes and accessory around for 10 years, and the com- Jivan also offers genome-wide proteins that modulate how and pany offers array products for both arrays for human, mouse, rat, fly, when a gene is spliced. basic research as well as for thera- and more, as well as targeted arrays peutic and diagnostic research. It for specific applications like oncol- Not perfect yet runs an internal research program ogy or toxicology. “Most of our for Alzheimer’s disease, measuring focus has been on microarrays until One problem with using micro- changes across many samples and more recently,” says Bingham, who arrays, of course, is that they can’t tissues. “You’ve got this capability of notes that because data analysis be used to find new variants since multiplexing on an array where poses such a problem, the company only known variants are spotted

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down. Even if the chip is meant to microarrays nowadays,” he says. cover the entire genome, not every While arrays do come with software splice variant is known for most analysis packages, none of them is genes, nor to what extent minor perfect; Penalva says his team, variants play a role in gene regula- which includes a bioinformaticist, tion. For disease genes, which tend usually has to try many different to produce splice variants in precise methods to get trustworthy data. percentages, finding minor variants “One thing that we observe when is especially important. you get array results, it looks like “In terms of looking at splicing, there are some probes there that are we’re fairly low tech right now,” simply not giving you any data, or says Lipscombe at Brown. “What data that doesn’t look correct,” we really need to know is whether Penalva says. “Sometimes … it’s a particular mRNA that encodes for better simply to discard this data.” a given isoform is present in suffi- Doug Black, a Howard Hughes cient quantity that we can say, ‘Oh, LUIZ PENALVA investigator at the University of yes, this must be meaningful.’ For microarrays should become stan- California, Los Angeles, uses a full sure we could use microarray,” she dard to replace the current arsenal of tools to study how splic- says, but correlating function usu- microarrays to characterize disease- ing is regulated and the role these ally comes back to isolating a vari- related changes and possibly even regulators play in neuronal cell dif- ant from one particular population for diagnostics,” he says. ferentiation. In mature neurons, he of cells. “Microarray analyses are Another limitation to using examines how calcium signaling probably better suited to scanning arrays is that even when a variant is pathways and chronic depolariza- a whole bunch of different tissues identified, it has to be validated tion can change splicing. While he and looking for differential distri- with PCR anyway and tested with incorporates both exon arrays and bution patterns, and so for us, functional assays. “I think, in gen- splice junction arrays into his day- because we know what we’re going eral, the technology is very, very to-day work, he says there are for — we’re just looking at one par- good — and what is important now tradeoffs to each. With exon arrays, ticular gene — the arrays are not is to apply it and understand how “[it] is a little bit of a pain and you necessarily better than what we’re these changes that are doing right now.” detected are actually being Most of the microarrays are based regulated in the cell and Determining which variants on EST database information, so which ones are relevant,” are actually being selected they tend to find large exons, Lip- Ule says. Determining scombe adds, ignoring possibly which variants are actually for and which are just errors critical drug target regions because being selected for and they vary by only a few amino which are just errors is an is an even bigger challenge acids. “I think there is an increase even bigger challenge to to the splice research in databases but there’s still a lot of the splice research com- information missing. Right now it’s munity at large. community at large. not refined” enough to discover A significant bottleneck tiny variations or variants in small for arrays is data analysis. Luiz end up having to validate a lot of or unique populations of cells, she Penalva, assistant professor at what you find through other says. Children’s Cancer Research Insti- means,” he says. However, when Jernej Ule of the MRC Laboratory tute at the University of Texas using exon junction arrays, plenty of Molecular Biology in Cam- Health Science Center in San of work has to go into probe bridge, UK, says he thinks most Antonio, says performing the design, and coming across new researchers use exon arrays since it analysis of his experiments, which variants isn’t possible the way it is would be too much bioinformatics attempt to identify splice variants with a genome-wide exon array. work to design an array that in glioblastoma, was the hardest Still, says Black, the splice junction probes exon junctions. But in part. “This is one of the major arrays are more sensitive and more coming years, “splice junction problems with alternative splicing reliable.

SEPTEMBER 2008 GENOME TECHNOLOGY 45

As a complementary tool, high- a little bit more high-throughput tered,” he adds. So far, he notes, the throughput sequencing holds than what people are using.” The most interest has been in arrays for much promise. Black is already approach allows him to look at cell surface and toxicology genes. using a Solexa sequencer to profile select splicing events across many In its work developing tools to splicing under different conditions. tissues and many conditions — study Alzheimer’s disease, ExonHit He believes that affordable next-gen experiments that probably wouldn’t began using splicing arrays as a sequencing will be able to take RNA be affordable using sequencing or diagnostic to screen patients for splice variant detection to the next microarrays. Whether PCR is used clinical trials. By measuring RNA level. “It’s potentially much more to validate microarrays or as a in circulating blood, scientists are accurate and much easier in the “replacement for cases where cus- able to determine whether some- analysis,” Black says. In fact, a tomers are looking at a particular one has Alzheimer’s, another form flurry of studies published recently pathway or a particular gene family,” of dementia, or a different disease used RNA sequencing, or RNA-seq, says Bingham at Jivan, it will con- altogether. Today the only way to to survey the complete transcrip- tinue to offer up more opportunities accurately pinpoint the disease is tomes of mice, Arabidopsis, yeast, in a multiplex setting. post-mortem, so splice signatures and human cells. “In practical have great potential in clinical terms, it’s not there yet,” Black Toward the clinic Alzheimer’s research. “The inter- adds. “You don’t get enough indi- esting thing about that signature is vidual short reads to sample all the As basic research moves ahead there are a lot of splicing isoforms exons that you want to sample. You with identifying new variants and that are present,” says Jaskowiak at don’t have enough sequence depth validating their functions in the ExonHit. “In many cases, it’s the cell, clinical research and ratio of isoforms that is impor- drug development are tant.” “ You don’t have enough already looking closely at Harvard’s Wolfe hopes that identi- how splice variants are fying splice variants that cause sequence depth without dysregulated in disease. Alzheimer’s will eventually be use- Often variants will be ful in finding effective drug targets. hundreds of thousands expressed alongside one Today, methods for slowing down of dollars to actually another, but in a healthy the progression of the disease percentage. When alter- mostly revolve around tweaking measure splice variants.” native splicing goes awry, gamma-secretase production, but one variant may be more gamma-secretase also plays a central or less expressed, leading role in the highly conserved Notch without hundreds of thousands of to signaling imbalances and dis- signaling pathway. Wolfe’s recent dollars to actually measure splice ease. It’s no surprise that finding work found that beta-secretase also variants.” drug targets would incorporate the undergoes alternative splicing and And in the event that you’re not study of splicing. could be an alternate drug target. By wading in funding, Sherbrooke’s “Initially, the most interest was interfering with its alternate splicing Benoit Chabot has come up with an coming from the academic world, events, one could interfere with affordable alternative to next-gen but what we’ve seen more recently beta-secretase function, he says. sequencing. As multiplexed PCR is that drug companies are starting “Alternative splice isoforms exist. takes off, Benoit has taken advan- to do pretty decent-sized studies,” They can vary depending on the cell tage of the capacity of newer says Jivan’s Bingham, “because type, and if we shunt splicing down machines to skip microarrays alto- splicing offers potentially more these alternative pathways, we can gether. Currently he can run 3,000 information for biomarkers than lower amyloid production.” RT-PCR reactions per day, and he gene arrays alone.” Splice arrays are Wolfe has also been studying the hopes to increase that 10-fold dur- used to look at expression signa- impact of splicing on tau, a gene ing the next few years. “We’re not tures for disease state or disease associated with a related, non- going to do microarrays,” says progression, to screen for specific Alzheimer’s form of dementia. He’s Chabot. “That means we’re not isoform drug targets, or to mark found that about half the known going global as much, but we’re drug response “where splicing mutations in the tau gene change going to automate it, make RT-PCR changes after a drug is adminis- its splicing “and somehow that

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leads to the self association of tau used 20 drugs on five different cules that would bind to disease- in clogging up neurons,” he says. cancer cell lines to see how they causing transcripts, effectively “What’s regulating the alternative affected apoptosis. While all the turning them off. Another might be splicing of tau? Can we pharmaco- drugs shifted splicing of Bcl-x in to make use of nonsense stop logically step in and tweak the the right direction, “it does not do codons, which in normal alternative system?” it systematically in all cell lines. splicing events tell the translation Discovering drugs that target one Some cell lines respond to it; other machinery to stop before the full- point in a splicing pathway can be cell lines don’t, depending on the length protein is complete. In the difficult considering the number of drug.” For other alternatively case of tau, the mRNA forms a tiny variables. A recent study out of spliced apoptotic genes, this wasn’t loop structure that Wolfe thinks Chabot’s Sherbrooke lab looked at the case — for some drugs it went could somehow be made to bind to how anticancer drugs affect splic- in the right direction and for oth- a small molecule to steer splicing ing of Bcl-x to promote apoptosis. ers it didn’t. “It’s very complex, toward one isoform or another. “If “It’s known that many types of and we cannot assume that taking there’s a structure, it probably has apoptotic genes are alternatively an anticancer drug will always go pockets where small molecules can spliced to produce pro-apoptotic in the right direction,” Chabot bind, so to the degree we can find variants or anti-apoptotic vari- adds. structure in the message that regu- ants,” Chabot says. To his surprise, There are a number of ways that lates splicing, we might identify he says, no one had ever systemat- alternative splicing events could be therapeutic targets to get very spe- ically looked to see if pro-apoptotic manipulated, all of which one day cific effects,” he says. “There are drugs actually initiated the apop- might be applied in the clinic. One some attempts at therapeutics, but totic pathway. In his study, he approach is to use antisense mole- it’s pretty early days.”

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SEQUENCING Upstream

INSEQUENCE cytochrome c oxidase I gene as a barcode. Sequencing Barcode of Life Project iBOL’s first aim is to Notes create a reference library by Leans on Sequencing barcoding 5 million speci- OXFORD NANOPORE mens representing TECHNOLOGIES acquired n interna- started last year at a work- 500,000 species within five exclusive rights to develop tional consor- shop at the University of years. This project, set to and market nanopore tech- tium of Guelph in Canada that begin next year, will signif- nologies from HARVARD Aresearchers brought together a variety icantly expand the current UNIVERSITY and collabo- says it plans to increase by of international researchers library, which comprises rators at the UNIVERSITY more than 10-fold the cat- with a shared interest in approximately 41,000 bar- OF CALIFORNIA, SANTA alog of eukaryotic species barcoding. coded species. CRUZ, and the NATIONAL that are tagged by a DNA Barcoding involves iBOL is currently focused INSTITUTE OF barcode, and to develop sequencing a short, stan- on raising at least $100 STANDARDS AND new barcoding technology dardized gene region that million of its $155 million TECHNOLOGY. The to identify specimens rap- differs between species. budget from various fund- company says label-free idly and inexpensively. In animals, for example, ing sources around the nanopore technology could The first phase of the researchers use a portion world, according to a proj- help to reduce the cost of project, which will gener- of the mitochondrial ect outline published by the DNA sequencing. ate a library of barcoded consortium in July. species, will largely What distinguishes this In its second-quarter earn- involve Sanger sequenc- DATAPOINT project most from other ings report, HELICOS ing technology, according large-scale efforts is that BIOSCIENCES said its to one of the organizers. each sequence read derives grant revenue was Second-generation from a different sample, $251,000 for the period, sequencing technologies according to Paul Hebert, while its net loss grew to will find applications in director of the Bio- $11.9 million, an increase environmental barcoding diversity Institute of Ontario of 47 percent from the studies later on, and a 2 at the University of Guelph same quarter in the previ- long-term goal of the proj- and an iBOL organizer. ous year. President STEVE ect is to develop a hand- HELICOS BIOSCIENCES To generate a 650-base LOMBARDI said problems held barcoding sequencer. ANNOUNCED RECEIVING read for each of hundreds with reagent stability The initiative, called A SECOND ORDER FOR ITS of thousands of samples, hindered efforts to get International Barcode of “Sanger sequencing tech- new sales. The company INSTRUMENT, BUT DID NOT Life Project, or iBOL, cur- nology is really the only fea- reported no revenue from rently involves 26 coun- NAME THE RESEARCH CEN- sible way to go,” he says. product sales during tries. Planning for iBOL TER PLACING THE ORDER. — Julia Karow the quarter.

FUNDED GRANTS

$577,448/FY 2008 $236,530/FY 2008 MOLECULAR ENGINEERING APPROACH TO STUDY MICROBIAL COMMUNITY PROFILING OF SEWAGE LONG-TERM SYNAPTIC PLASTICITY CONTAMINATION IN THE GREAT LAKES Grantee: Jingyue Ju, Columbia University Grantee: Sandra McLellan, University of Wisconsin Began: Feb. 1, 2008; Ends: Jan. 31, 2012 Began: Jun. 15, 2008; Ends: May 31, 2010 Ju and his team will continue development work on McLellan and her team will use massively parallel their Massive Parallel DNA Sequencing Chip System DNA sequencing strategies to study the microbial for use in digital gene expression, with the aim of doing communities and other sewage contamination present large-scale expression studies in single neurons. in the Great Lakes in the northern US. The lakes serve According to the abstract, the tools will be tested on as drinking water to 40 million people, and bacterial the memory-forming network of Aplysia, a unique content of the water is unknown. model organism for neurobiology.

SEPTEMBER 2008 GENOME TECHNOLOGY 49

PROTEOMICS Upstream

PROTEOMONITOR found 18 proteins associated with Alzheimer’s. Proteomics Satoris to Launch In that study, researchers Notes looked at 259 archived Alzheimer’s Test blood samples ranging The Proteomics from patients who had no Research Group of hile pro- protein panels within the symptoms to those who the ASSOCIATION teomics next few months, says had advanced Alzheimer’s. OF BIOMOLECULAR holds Cris McReynolds, presi- The resulting 18-protein RESOURCE FACILITIES W promise dent and chief executive panel had both sensitivity is recruiting volunteers as a technology that officer of Satoris. and specificity of about 90 to be part of a study could potentially help in Both panels are based on percent and was able to looking at different ways the early diagnosis of work published in the fall “pick out the Alzheimer’s to determine quantitative Alzheimer’s disease, no in Nature Medicine,in from a population of differences in several one has yet been able to which researchers, includ- dementia and to properly proteins in six human translate proteins into a ing those from Satoris, identify those who had plasma samples. predictive tool for the AD,” according to debilitating ailment. McReynolds. MIRACULINS will buy a DATAPOINT But a small molecular Authors of the study also portfolio of biomarkers diagnostics firm, Satoris, say that the panel was able from Toronto’s MOUNT is trying to change that. Its to identify patients with SINAI HOSPITAL to chief executive says the mild cognitive impairment develop a diagnostic for company’s protein-based $ who eventually were diag- pre-eclampsia. Miraculins Alzheimer’s test may be nosed with Alzheimer’s recently shifted its focus ready to hit the market by two to six years later, but from proteomics research the end of the year. McReynolds says it is not and development to diag- 90BILLION The company is cur- clear whether that means nostics development. rently collaborating with the panel is predictive of the Mayo Clinic to vali- ANNUAL the disease or that it is only GEORGE MASON date results from a study “able to detect the early dis- UNIVERSITY scientists REVENUE FROM published late last year. ease process associated partnered with FAIRFAX- The validation work is GENERAL ELECTRIC’S with AD.” Though mild NORTHERN VIRGINIA expected to be completed TECHNOLOGY cognitive impairment is HEMATOLOGY in September and, INFRASTRUCTURE UNIT, believed to be a possible ONCOLOGY to depending on the results, precursor to Alzheimer’s, determine the protein WHICH WILL NOW it could clear the way for some patients with it do signaling pathways Satoris to hit the market INCLUDE not develop Alzheimer’s. involved in multiple with two Alzheimer’s GE HEALTHCARE. — Tony Fong myeloma.

FUNDED GRANTS $46,826/FY 2008 $78,500/FY 2008 NOVEL MASS SPECTROMETER FOR COMPREHENSIVE INTRACELLULAR MYCOBACTERIAL PROTEOME NO-LOSS MS/MS OF ALL STORED IONS Grantee: Qingbo Li, University of Illinois at Chicago Grantee: Sunnie Myung, Rockefeller University Began: Apr. 1, 2008; Ends Mar. 31, 2009 Began: Jan. 1, 2008; Ends: Dec. 31, 2010 Li plans to investigate the proteome of M. tuberculosis Myung plans to optimize high-capacity ion trap mass H37Rv found within human and murine macrophages spectrometers by isolating the pressure within the ion using liquid chromatography/linear ion trap-Fourier trap and changing the geometry of the trap to increase transform mass spectrometry. Then he will compare resolution. Furthermore, she will couple an orthogonal those proteomes to identify the active intermediary injection reflectron TOP mass analyzer to the high- metabolism pathway, particularly focusing on the capacity ion trap. Then she will use the instrument to half tricarboxylic acid cycles. Li says this may help study abnormal levels of protein phosphorylation in researchers understand the intracellular persistence cancer, diabetes, and rheumatoid arthritis. of mycobacteria.

SEPTEMBER 2008 GENOME TECHNOLOGY 51

The siRNA breakthrough that changes the world of RNAi.

140 RNAi research is no longer constrained by cell types that are resistant Target mRNA Levels 120 Cell Viability to conventional lipid-based delivery reagents. Thermo Scientiﬁc 100 ® ™ Dharmacon Accell siRNA provides a breakthrough in siRNA delivery. 80

It is specially modiﬁed for delivery into any cell type, without the 60

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Moving science forward

RNAi Upstream

RNAiNEWS Tacere is exploring are hinted at by two recent RNAi Notes Tacere Evaluates New additions to the company’s staff. While both have ISIS PHARMACEUTICAL Drug Opportunities experience with RNAi, is conducting a phase II “one has more of a [neurol- trial of mipomersen, its aving found “Some [possibilities] we ogy] background and one antisense-based drug to licensees for are still looking at [and] has an immunology back- treat heterozygous familial its lead some we’ve passed on,” ground,” Hall says. Catelani hypercholesterolemia. Hexpressed says Mike Catelani, Tacere notes that Tacere now has GENZYME exclusively RNAi drug, the preclinical chairman, president, and six full-time employees. licensed rights to the drug. hepatitis C therapy TT- CFO. “We’re not in a posi- Despite Hall’s reticence, 033, Tacere Therapeutics tion at this point to talk she notes that Tacere ALNYLAM PHARMA- continues to seek its next about anything we’re expects to find its next CEUTICALS exclusively pipeline candidate. looking at, but there are pipeline program outside licensed intellectual prop- According to Tacere some interesting things of its own labs and that it erty to RNA activation, a CEO and co-founder Sara out there.” will likely remain focused gene up-regulation tech- Hall, the company is in But Hall says the fields on expressed RNAi. nology. The agreements “evaluation mode” and “We don’t really want to are with UNIVERSITY OF has been in discussions start over again like we did TEXAS SOUTHWESTERN with a number of undis- DATAPOINT with TT-033 because MEDICAL CENTER; the closed parties, both from that’s a long, hard road,” UNIVERSITY OF academia and industry, Hall says. “We’d like to CALIFORNIA, SAN about potentially in- pick up something that’s at FRANCISCO; and the licensing new programs. the translational research SALK INSTITUTE FOR “We had some really good stage.” BIOLOGICAL STUDIES. meetings at [the Biotech- TT-033 was originally nology Industry Organiza- developed by Avocel, a PFIZER began a phase II tion’s international meeting 2009 company Hall co-founded, trial of PF-4523655, an in San Diego] and we’ve which was acquired by siRNA-based drug licensed done some pretty extensive Australian expressed RNAi from QUARK PHARMA- due diligence on a couple THE YEAR DURING firm Benitec in 2004. Fol- CEUTICALS, in patients things,” she says. WHICH SIRNAOMICS lowing a sweeping corpo- with diabetic macular Still, the search is early- PLANS TO BEGIN A PHASE I rate reorganization, Ben- edema. It targets a stage, and Hall declined to itec licensed the drug’s gene involved in angio- TRIAL OF ITS MULTI-SIRNA comment in detail on the worldwide rights to Tacere genesis, vascular perme- indications or therapeutic COCKTAIL FOR OCULAR in late 2006. ability, and retinal areas Tacere is considering. DISEASES — Doug Macron neuron death.

FUNDED GRANTS

$229,600/FY 2008 $258,052/FY 2008 NOVEL TUMOR SUPPRESSOR GENE DISCOVERY IN INDUCIBLE RNAI IN SPERMATOGONIAL STEM CELLS PANCREATIC CANCER Grantee: Jon Oatley, Pennsylvania State University Grantee: James Eshleman, Johns Hopkins University Began: Jun. 1, 2008; Ends May 31, 2010 Began: Sep. 1, 2007; Ends Aug. 31, 2009 With this grant, Oatley will be seeing if RNAi can lead to Eshleman will be using RNAi-based techniques to find advances to treat male infertility. He plans to determine novel pancreatic cancer tumor suppressor genes. First, the efficacy of vector-based RNAi on silencing gene he will transduce non-tumorigenic and weakly tumori- expression in spermatogonial stem cells, as well as to genic pancreas cell lines with an RNAi library and grow evaluate the Tet-On system for inducing RNAi in those them up in agar and in nude mice. Then, he will select cells, and, finally, to determine the efficacy of inducible tumorigenic cell clones and sequence, test, and RNAi to silence essential spermatogonial stem cells’ validate the RNAi. self-renewal genes.

SEPTEMBER 2008 GENOME TECHNOLOGY 53

______

BIOINFORMATICS Upstream

BIOINFORM encoded in the XML file that describes images that Bioinformatics Scientists Debate are part of a publication. Notes “That makes the parsing of Publishing Future a figure easier,” he said. BIOMAX INFORMATICS Yale’s Mark Gerstein will integrate a toxico- t the Intelligent meantime it will be impor- favors the idea of linking genomics database Systems for tant to find ways to databases and journal arti- developed by the Molecular Biol- “improve practices of cles so that scientists can MOUNT DESERT Aogy conference, defining content,” which track a given gene annota- ISLAND BIOLOGICAL researchers weighed in on would make text- and tion in a database back to LABORATORY with its the role that bioinformatics image-mining easier. the published paper. data-management plat- tools will play in the future of One notion he described “There is no good frame- form. The MDI database scientific publishing. is the idea of “structured work for browsing through system includes informa- During the conference, digital caption” that would the genome in the frame- tion about cross-species several speakers discussed not show up in the printed work of publications,” he interactions between new text-mining tools and paper but would be said. While loading that genes, chemicals, and other methods for extract- information into one proteins that can be ing information from scien- monolithic database may used to study disease DATAPOINT tific papers. For example, not be possible, federated susceptibility and diseases Carnegie Mellon’s Robert queries across structured, that are influenced by Murphy spoke about his ontology-oriented abstracts the environment. work on parsing informa- could help, he suggested. tion about images from the $ He said his group “is very SIMULATIONS PLUS has biological literature. He keen on this idea of struc- signed a multi-year collabo- and his colleagues have tured abstracts” as a small ration with ROCHE, which developed SLIF, Subcellular 3 start to enable a connection will provide funding and Location Image Finder, a MILLION between journal articles feedback in developing the platform that can extract and databases. Since firm’s GastroPlus software information from captions authors already write the program. Simulations Plus COMPUGEN’S NET LOSS and figures containing flu- abstracts, this concept will collaborate with Roche orescence microscopy FOR THE QUARTER ENDING would ensure the high scientists to advance the images. JUNE 30, 2008, INCLUDING quality of the machine- capabilities of GastroPlus Murphy stated in his A NON-CASH EXPENSE readable abstract. Those to simulate drug-drug inter- presentation that he texts in turn could be the actions. Roche is slated to OF $416,000 RELATED believes these types of sys- training set for a more provide funding for the tems will become more TO STOCK-BASED large-scale machine reading equivalent of one full-time widespread but in the COMPENSATION. project. — Vivien Marx scientist for two years.

FUNDED GRANTS

$1,200,000/FY 2008 $273,906/FY 2008 EDAC: ENCODE DATA ANALYSIS CENTER ADAPTIVE PERSONALIZED INFORMATION MANAGEMENT Grantee: Ewan Birney, European Bioinformatics Center FOR BIOLOGISTS Began: May 15, 2008; Ends: Mar. 31, 2012 Grantee: William Cohen, Carnegie Mellon University This proposal aims to facilitate the integration of data from Began: Jul. 11, 2008; Ends: May 31, 2012 multiple sources using sophisticated statistical models This funding will enable the development of an adaptive and machine learning techniques to build integration information management tool. Cohen and his team intend methods combining datasets. Birney and his team will also to exploit recent advances in machine learning and data- use this grant to provide quality assurance and summary base systems in order to facilitate their scheme for loosely metrics of genome-wide multiple alignments. Overall, they integrating both structured information and unstructured aim to provide deep integration of the ENCODE data, text, and then querying the integrated information using under the direction of the AWG and in tight collaboration easily formulated similarity queries. with the other members of the ENCODE consortium.

SEPTEMBER 2008 GENOME TECHNOLOGY 55

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MICROARRAYS Upstream

BIOARRAYNEWS Santa Clara, Calif.-based company the opportunity Microarray For WTCCC, Agilent Will to iron out the protocol for Notes high-throughput, auto- Provide Custom Chips mated array processing. DARPA has granted “We expect that enhance- COMBIMATRIX $250,000 gilent Tech- Gene Technology, an ments to the comparative for proof-of-concept nologies will Agilent certified service genomic hybridization research to find new uses provide cus- provider, will use Agilent’s workflow demonstrated in for the company’s micro- Atom, whole- Velocity 11 Bravo robot to this project, such as the use array technology, including genome copy number vari- run the samples at OGT’s of the Velocity 11 Bravo label-free detection sys- ation-focused arrays to the lab. System and the use of a tems for use in diagnostics, Wellcome Trust Case Con- For Agilent, the high- plate for purification, will chemical measurement, trol Consortium, which volume deal will enable it to facilitate widespread use of and chemical agent will use them in the second hone a CNV-focused Agilent CGH/CNV micro- detection. phase of its 19,000-sample microarray product line arrays in high-throughput study to identify genetic scheduled to launch later environments,” says ASPER BIOTECH,an variants influencing dis- this year. It also gives the Yvonne Linney, Agilent’s Estonian biotechnology ease susceptibility in a vari- vice president and general company, has obtained a ety of rare and common manager of genomics. license to commercialize DATAPOINT diseases. “Agilent also plans to pro- a new method for SNP- Specifically, the consor- vide a very high-resolution genotyping called arrayed tium will attempt to link CNV-focused catalog array primer extension-2 from genes to tuberculosis, coro- with 1 million features the ESTONIAN nary heart disease, types 1 $ using data from public BIOCENTRE. and 2 diabetes, rheumatoid databases and Agilent’s own arthritis, Crohn’s disease, and collaborative CNV Iowa-based INTEGRATED bipolar disorder, autoim- 342 research,” she says. DNA TECHNOLOGIES has mune thyroid disease, MILLION Matthew Hurles, a geneti- completed the expansion of ankylosing spondylitis, cist at Wellcome Trust, said its 21,500-square-foot multiple sclerosis, breast in a statement that the European oligonucleotide cancer, and hypertension. ILLUMINA RAISED WTCCC “aims to character- production facility in As part of its deal with the $342.6 MILLION ize [the] most common Haasrode Research Park in group, Agilent will design IN PROCEEDS structural modifications of Leuven, Belgium. The facili- and fabricate the custom DNA that may play a ty will allow it to provide FROM ITS PUBLIC CNV chips, printing two causative role in these better services for its cus- arrays of 105,000 probes STOCK OFFERING diseases.” tomers in Europe, the each per slide. Oxford THIS AUGUST. — Justin Petrone Middle East, and Africa.

FUNDED GRANTS $1,199,769/FY 2008 $291,274/FY 2008 MICROARRAY CENTER FOR RESEARCH ON THE UNIVERSAL, COMPACT COMBINATORIAL MICROARRAYS NERVOUS SYSTEM FOR DNA BINDING SITE DISCOVERY Grantee: Dietrich Stephan, TGEN Grantee: Martha Bulyk, Brigham and Women’s Hospital Began: August 1, 2005; Ends: May 31, 2010 Began: July 26, 2006; Ends: June 30, 2009 This Affymetrix Center of Excellence offers a platform In this project, Bulyk will develop the use of compact com- to conduct research on the nervous system using binatorial DNA microarrays in protein-binding microarray microarrays. TGEN will provide assistance with experi- experiments in order to identify all possible DNA binding mental design, data generation, data interpretation, sites of sequence-specific transcription factors. She will and data dissemination. New research goals include also determine the binding affinities of all possible DNA having scientists submit tutorials and projects online, binding sites for 15 S. cerevisiae transcription factors, as review by an expert IRC, and data warehousing with a well as evaluate the utility of binding affinity data for six-month timed release to the public. improved prediction of in vivo binding sites.

SEPTEMBER 2008 GENOME TECHNOLOGY 57

© 2008 Thermo Fisher Scientific Inc. All rights reserved. Part ofThermoFisherScientific Stand out fromthebackground [email protected] www.thermo.com/microarray for spot-on results. Make sureall your results stand Choose out. SuperChip AB and provide areliable surface for your proteinstudies. SuperChip ABis theclear choice toreduce background binding properties you demand for your research. proprietary microarray surface, provides thecovalent protein-friendly substance that, whencombined with our Already familiar toscientists, agarose is an economical, reduces thebackground that can with signal interfere detection. features aproprietary agarose-based surface that significantly Scientific microarray substrate forproteomics anddiscovery, low abundance proteins. SuperChip™ AB,thenew Thermo It’s nosecret that background noise drowns out signals from ______rvosPg otns|Zo n|Zo u rn oe erhIse|Nx Page Next | Issue Search | Cover Front | out Zoom | in Zoom | Contents | Page Previous Page Next | Issue Search | Cover Front | out Zoom | in Zoom | Contents | Page Previous quality while reducing background. applications, enhance binding microarray substrates SuperChip ABagarose-based forproteomic A A B B E E F F M M a a G G S S A Genome Technology Previous Page | Contents | Zoom in | Zoom out | Front Cover | Search Issue | Next Page BEF MaGS

PGx & MOLECULAR Dx Downstream

PHARMACOGENOMICSREPORTER is FDA-cleared for CUP, and its regulatory approval PGx & Molecular Pathwork First to Net also marks an achievement Dx Notes for the agency. The Tissue FDA OK for CUP Dx of Origin test is only the Northern Ireland-based second in vitro diagnostic ALMAC DIAGNOSTICS y being the which Agendia markets in multivariate index assay to kicked off an international, first to Europe as CUP-Print. receive the FDA’s approval. multi-center collaborative launch an AviaraDx plans to submit The FDA has said that it lung cancer study aimed at BFDA- and its test for FDA clearance in intends to regulate so- finding a prognostic gene CLIA-approved test the next 12 to 18 months. called IVDMIAs, a subset expression signature for designed to help physi- Meanwhile, Rosetta of laboratory-developed patients with early non- cians determine cancer Genomics and Exiqon are tests that combine multiple small cell lung cancer. type in a tumor, Pathwork both developing a diag- variables into a single, pre- The study will be led by Diagnostics may have nostic for the indication. dictive test, and has issued Dean Fennell at QUEEN’S gained a significant advan- However, Pathwork’s two draft guidances in this UNIVERSITY BELFAST. tage over competitors. product is the only test that regard. The first IVDMIA to This summer, Pathwork receive a nod from the HX DIAGNOSTICS received 510(k) clearance FDA was Agendia’s expanded a license and from the FDA for its Tissue DATAPOINT MammaPrint assay for development agreement of Origin test. breast cancer recurrence. with NANOGEN, under The test, which uses According to the which the companies will Pathwork’s PathChip National Cancer Institute, develop rapid, point-of-care microarray and runs on $ between 2 percent and 4 diagnostics for respiratory Affymetrix’s GeneChip percent of US cancer infectious diseases. The platform, “is the first cus- patients have a cancer for firms are developing a fluid tom Affymetrix gene 27.8 which the primary site is test to detect multiple expression array to be MILLION never identified. David strains and subtypes cleared for diagnostic Craford, Pathwork’s VP of of influenza, including use,” the FDA noted in a commercial operations, avian influenza, GENOMIC HEALTH statement. says that “the cost to in one test. Pathwork is not the only REPORTED THAT SECOND- identify a primary tumor company with a diagnostic QUARTER REVENUE WAS can be significant,” since SEQUENOM certified its test for cancer of unknown $27.8 MILLION, UP 89.1 traditional treatment first Center of Excellence for primary origin. Currently, approaches involve run- genotyping technology — PERCENT FROM ITS AviaraDx markets a CUP ning multiple diagnostic the MCGILL UNIVERSITY assay in the US, called REVENUE IN THE SAME technologies in parallel. and GENOME QUEBEC Aviara CancerTYPE ID, QUARTER LAST YEAR. — Turna Ray Innovation Centre.

FUNDED GRANTS

$658,595/FY 2008 $572,928/FY 2008 PROTEOME SIGNATURES AND TARGET VALIDATION IN CORTICOSTEROID PHARMACOKINETICS AND LYMPHOMAS PHARMACODYNAMICS Grantee: Daniel Jay, Tufts University Grantee: William Jusko, SUNY Buffalo Began: May 1, 2004; Ends: Jul. 31, 2010 Began: Jul. 1, 1977; Ends: Jun. 30, 2010 Jay and his colleagues will use the funds to improve the Currently, this NIGMS grant is funding Jusko and his col- use of proteomics as a pharmacogenomic tool by devel- leagues to study pharmacogenomics as well as pharma- oping surface proteome signatures and performing cokinetics and pharmacodynamics in rat. The team will functional proteomic target validation analysis directly study genes from liver, muscle, and kidney tissue using on primary tumor tissue. Part of the proteome signature microarrays, and hopes to assess the expression of bio- will help differentiate how someone is responding to a markers for diabetes. therapeutic.

SEPTEMBER 2008 GENOME TECHNOLOGY 59

Downstream CASE STUDY

Diagnosing the Unknown It’s hard enough to treat cancer — but it’s more difficult when the primary tumor can’t be found. Researchers aim for molecular diagnostics to decipher the mystery of CUP. By Ciara Curtin

s a clinician, Gauri “In the era of molecular diagnostics, spares patients additional biopsies Varadharachary has it’s the right time to define and for fresh sample. seen quite a few select subtypes,” she says. In partic- Validating that those subtypes patients with ular, the team is using immunohis- are what they suspect, though, is a Ametastatic tumors tochemistry and RT-PCR panels. challenge. By definition, no one for which no amount of tests or In a recent Lancet Oncology paper, knows from what tissue the CUP imaging could determine the pri- Varadharachary reports that she’s tumor came. “Validation is indi- mary tumor site. If after CT scans, been able to parse out a subtype of rect,” Varadharachary says. She PSA testing, mammograms, and CUP based upon its immunohisto- adds that it is based on how long more targeted testing, the site still chemistry. By looking at the cyto- the patients live, how aggressive remains elusive, then the patients keratins on the tumor cell surface their cancer is, and how they react are diagnosed with carcinoma of — which aren’t altered as a cell to a tailored treatment, if one unknown primary and are often transforms from normal to malig- exists. If the tumor has the mark- treated with systemic chemother- nant — Varadharachary can trace ings of a colon cancer and also acts apy instead of targeted therapies. the cell’s lineage back to before it like it, then it probably is colon Varadharachary, though, is working became a tumor. When a tumor has cancer. to change that by developing a CK20+/CK7- immunostain pro- But will molecular diagnostics be molecular-based methods to deter- file and is positive for CDX2, a able to determine CUP tissue of ori- mine the origin of these types of nuclear transcription factor and gin? “It’s still early to say,” Varad- tumors. product of a homeobox gene that harachary says. The tests will work, These kinds of cancers are hetero- promotes intestinal differentiation, she says, but not alone. “There geneous metastatic tumors. The Varadharachary calls this colon- won’t be one gold standard,” she incidence of CUP is estimated to be cancer-profile CUP. The patients says; molecular diagnostics will between three and five percent of all with this profile, she says, benefit have to be used in conjunction with cancers, about the same percentage from colon-cancer-based treat- imaging and immunohistochemical as pancreatic cancer. The American ments — a sign that it really was a assays. Part of the ongoing project is Cancer Society says that only half of metastatic colon tumor. to assess how the RT-PCR molecu- CUP patients live nine to 12 In addition to immunohisto- lar diagnostic correlates with the months after their diagnosis. A chemistry, Varadharachary is also immunohistochemistry results. 1994 study from the MD Anderson working on a molecular assay to In the end, determining the CUP’s Cancer Center’s James Abbruzzee, diagnose CUP subtypes. She and original tissue comes down to in whose group Varadharachary her colleagues are developing a patient care. By knowing someone now works, reported 10 to 15 per- prospective trial of a 10-gene RT- has colon cancer rather than breast cent of CUP patients lived at least PCR panel. They’re including cancer, there are different therapies five years after diagnosis. colon-specific marker cadherin-17, to give the patient a better chance of Recently, Varadharachary and her which is also expressed in both nor- survival. But there’s a dearth of tai- colleagues in the Abbruzzee group mal and cancerous colon tissues. lored treatments for many cancers, have started focusing on determin- They will then be testing the panel such as pancreatic cancer, Varad- ing molecular profiles of subsets of on formalin-fixed paraffin-embed- harachary says. When that CUP so that a tailored treatment can ded tissue samples from CUP changes, treating CUP patients will be developed to better treat them. patients — a technical feat that improve as well.

60______WWW.GENOME-TECHNOLOGY.COM SEPTEMBER 2008

Q&A: GURVANEET RANDHAWA Downstream

Too Much Information Clinical genomics advisor Gurvaneet Randhawa on evidence-based medicine and the ‘information overload’ that’s making it tough to translate research into patient care.

urvaneet Randhawa fashion that is digestible and use- is the senior advisor able in the clinic. To do all of those on clinical genomics steps — specifying what questions and personalized you want to answer, collecting all of Gmedicine at the the research evidence in a system- Agency for Healthcare Research and atic fashion, and integrating that Quality, a unit within the US Depart- knowledge with expertise and ment of Health and Human Services. patient values — requires a multi- GT’s Jeanene Swanson caught up with disciplinary team of people working him to discuss the use of evidence- on these reports for several based medicine in the clinic, and how months. far along systems biology tools have The challenge we are struggling come in affecting patient care. with is how to do it more consistently and uniformly, and one of the GENOME TECHNOLOGY: What is options is to have available different evidence-based medicine? credible sources of information that GURVANEET RANDHAWA can guide the clinicians. An example GURVANEET RANDHAWA: Evi- of that are the recent recommenda- GR: Getting good, validated infor- dence-based medicine typically tions released by the US Preventive mation is the first challenge. It’s implies three different elements that Services Task Force on prostate can- important to have large-scale stud- need to be considered together, the cer screening. ies, but ... there are many other fac- first being the best and current sci- tors beyond genetics and beyond entific evidence, the second being GT: How does large-scale biological markers that influence the causation the clinical expertise, and the third research play a role here? of disease. being the patients’ values — combin- The second challenge is, even if we ing all of these in making decisions GR: The task force hasn’t ventured know what genes or what biological jointly between the clinician and the too much in that area because it deals factors are predictive of what dis- patient in terms of what is the best mostly with clinical prevention. eases, what do we do with that infor- course of action to take. AHRQ’s mission is to improve the mation? There is no drug that is with- effectiveness, safety, quality, and out adverse events. We have to assess GT: How do you apply that in clini- efficiency of healthcare. So our the balance of benefits and harms. cal practice? focus is on things that are already The third challenge is trying to being used in clinical practice, or implement the best possible GR: That is a very challenging that are new to clinical practice and research evidence into practice, aspect, primarily because given the still haven’t gained widespread use. and again, there’s a huge informa- workload, there really isn’t enough All of the ’omics studies tend to be tion overload that is occurring. time for the busy primary care more exploratory analyses. The information needs to be reli- physician to look up all the latest able and credible, and you have to studies that are being published, let GT: What are some of the challenges make sure that it is interoperable alone try and synthesize them in a facing evidence-based medicine? across different systems.

SEPTEMBER 2008 GENOME TECHNOLOGY 61

Events MEETINGS AND DEADLINES

Conferences DATE CONFERENCE ORGANIZER LOCATION CATEGORY Sep 1-3 Protein Function Prediction Tools EBI-ENFIN Hinxton, UK Bioinformatics Workshop Sep 1-4 11th International Meeting of MGED Microarray and Gene Riva del Garda, Arrays Expression Data Society Italy Sep 2-6 Metabolomics 2008 Metabolomics Society Boston Metabolomics Sep 7-10 AIRI Annual Meeting Association of Independent Washington, DC General Research Institutes Sep 10-14 Genome Informatics CSHL/Wellcome Trust Hinxton, UK Bioinformatics Sep 16-18 RNAi Europe Select Biosciences Stockholm, RNAi Sweden Sep 17-18 Advances in qPCR Select Biosciences Stockholm PCR Sep 22-23 Biosimilars2008 Scherago International Washington, DC Clinical Sep 24-25 Exploring Next-Generation Sequencing CHI Providence, RI Sequencing Sep 25-28 The Pathologists' Meeting College of American San Diego Clinical Pathologists Sep 27-30 HGM 2008: Human Genome Meeting HUGO Hyderabad, India Genomics Sep 29 - Oct 1 Biomarker Discovery Summit CHI Philadelphia Biomarkers Oct 9-12 Personal Genomes Cold Spring Harbor Cold Spring Translational Laboratory Harbor, NY Oct 12-17 International Biotechnology IUPAC Dalian, China General Symposium & Exhibition Oct 14-17 NIH Research Festival NIH Bethesda, Md. General Oct 16-17 Personalized Health Care National Ohio State University Columbus, Ohio Translational Conference Oct 20-22 Discovery2Diagnostics IBC San Diego Translational Oct 20-24 Discovery on Target CHI Boston Pharma Oct 22-24 Northeast Regional Life Science Burlington, VT Core labs/ Core Directors Meeting instrumentation Oct 30 - Nov 2 AMP 2008 Assocation of Molecular Grapevine, Texas Clinical Pathologists Nov 1-5 Mass Spectrometry Applications University of California, San Diego Proteomics to the Clinical Laboratory San Diego Nov 10-11 Burrill Personalized Medicine Meeting Burrill & Company San Francisco Personalized medicine Nov 10-13 qPCR Symposium USA IES / TATAA Millbrae, Calif. PCR Nov 11-15 ASHG 2008 American Society of Philadelphia Genomics Human Genetics Nov 13-14 Personalized Medicine Conference Harvard Partners Center Boston Personalized medicine Nov 15-19 Neuroscience 2008 Annual Meeting Society for Neuroscience Washington, DC Neuroscience Dec 13-17 American Society for Cell Biology ASCB San Francisco General Annual Meeting 2009 Jan 5-9 Pacific Symposium on Biocomputing Kohala Coast, Bioinformatics Hawaii Jan 10-14 Plant and Animal Genome Meeting XVII Scherago San Diego Genomics Jan 11-16 PepTalk CHI San Diego Proteomics Feb 4-7 Advances in Genome Biology and Gcorp Marco Island, Fla. Genomics Technology Feb 7-10 ABRF 2009 Association of Biomolecular Memphis, Tenn. Core facilities Resource Facilities Feb 12-14 AUTM 2009 Association of University Orlando Tech transfer Technology Managers Feb 22-25 Fifth Annual US HUPO US HUPO San Diego Proteomics Feb 24-27 Molecular Medicine Tri Conference CHI San Francisco Translational

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Deadlines

SEPTEMBER 8 and development of com- Application deadline mercializable biomarker for NIH’s SHARED technologies relevant to NEUROBIOLOGY OF mental disorders. FRAGILE X SYNDROME AND AUTISM grant, which SEPTEMBER 22 will award funds to study Application deadline for the neurobiology of the CISE COMPUTING patients with both FXS RESEARCH INFRA- and autism and to identify STRUCTURE program. novel drug targets for This NSF award will fund these diseases. the creation, enhancement, and operation of world- SEPTEMBER 8 class computing research Application deadline for the infrastructure. DIRECTED STEM CELL DIFFERENTIATION FOR SEPTEMBER 25 CELL-BASED THERAPIES Application deadline FOR HEART, LUNG, AND for the NIH award for BLOOD, AND AGING APPLICATION OF DISEASES grant. This NIH EMERGING TECH- award is for researchers to NOLOGIES FOR study the differentiation of CANCER RESEARCH. embryonic or adult stem or This grant will fund the progenitor cells, either in development of tech- vitro or in vivo. nologies, including tools and instrumentation, for SEPTEMBER 8 cancer-relevant molecular Application deadline for analyses in vitro, in situ, the PILOT STUDIES IN and/or in vivo. This grant PANCREATIC CANCER is part of the broader NCI- grant. This funding from sponsored Innovative NIH will support multi- Molecular Analysis disciplinary research of Technologies program. pancreatic cancer, including studies on genetic SEPTEMBER 25 causes, biomarkers, and Application deadline for the identification of new the INNOVATIVE TECH- drug targets. NOLOGY SOLUTIONS TO CANCER SAMPLE SEPTEMBER 8 PREPARATION grant. ______Application deadline for This NIH award, also part of the DEVELOPMENT OF the NCI’s IMAT program, BIOMARKERS FOR will support technology MENTAL HEALTH development in preparing, RESEARCH AND purifying, processing, and CLINICAL UTILITIES handling cancer-relevant grant from NIH. This samples for molecular award supports research analyses. ______

Lab Reunion THE ALTMAN LAB

A Man of Many Hats Russ Altman is many things to many people: researcher, physician, academic advisor. He’s never lost sight of his passion for science and the high expectation he places on himself and those who pass through his lab. By Matthew Dublin

s an MD/PhD, Russ learning, and natural language PhD, but then when they hit the Altman is a member processing methods to conduct real world, they have not done this of that rare breed of protein and RNA structure analysis. activity that the world is expecting individuals who And as principal investigator for the them to do.” A really do have one NIH Center for Biomedical Compu- foot planted firmly at the bench tation at Stanford, Altman helps What they’re made of and the other in the clinic. Upon develop software tools to simulate finishing his undergraduate studies biological systems in terms of the Altman says that his grad students in biochemistry at Harvard Univer- motions of their components. really get to see what they’re made sity in 1983, he headed off to Stan- In addition to dual roles of of in year two or three of their grad- ford University, initially thinking researcher and physician, Altman uate studies. “They have done the he would pursue graduate work in is also a mentor and advisor to confidence builder projects, maybe biophysics. But when he arrived, many. He says his approach to they’ve been on one paper as a mid- he heard about the university’s advising is based upon what he dle author, but now they have to medical information sciences pro- considers to be an “old fashioned” commit themselves to three or four gram that appealed to his twin interpretation of the PhD as a straight years working very deeply interests of computer science and hunting license to define and solve on a problem and defining what it biology. Years later, when Altman a problem independently. Along is,” he says. “It’s hard, and I want took over as director of the pro- those lines, he tries to stay as much them to be primarily responsible for gram in 2000, he renamed it the out of his students’ biomedical informatics program. way as possible dur- “I try not to be too directive Also the chair of Stanford’s bioengi- ing the early stages of neering department, Altman con- their journey to a in the very beginning stages tinues to maintain a small medical doctorate. “I try not practice despite juggling his many be too directive in the where they’re looking for their research projects. very beginning stages thesis project.” Altman’s lab is currently focused where they’re looking on developing PharmGKB, a phar- for their thesis project. I have a ton it. And they are very stressed out, macogenomics database that aims of ideas but I really feel it’s impor- more than in any other time.” to be the Web destination for tant for them to articulate what the Sometimes, he must act as a life pre- information about genes that are problem is, why it’s important, server for a student by throwing in important for drug response. and how to go after it,” Altman his own ideas about the direction “We’re building this database and says. “I work with them on all of their research should take, but this we’re doing informatics research on those things, but it would be too rarely yields the desired outcome of how to deliver services and how to easy for me to feed them the prob- producing an independent and analyze data relevant to pharma- lem and why it’s important — and mature investigator. cogenomics,” he says. He also then they would just be techni- Pressure and dedication take on a directs Stanford’s Helix Group, cians implementing the work. … whole new meaning for those which uses simulation, machine That might be fine for getting a brave souls endowed with the

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desire to embark on the MD/PhD cally the same for everybody. At the to completely reorganize the struc- path to which Altman was drawn. end of the day, everyone takes the ture of the grant,” says Laederach. He has some advice for those con- same nationalized exam, so the “It was exactly what the grant sidering it: “The first thing I say to quality of medical school training is needed, but it took a lot of guts to somebody like that is, ‘You have to pretty much even,” says Altman. start cutting and pasting everything want to be a doctor to go to med- “But the quality of PhD training is in such a major way three days ical school, and you have to want to highly variable. Given that, I before the due date. But we ended be scientist to go to PhD school,’” he would choose your program based up getting it funded.” says. “What I mean by that is: do on offering first-class PhD creden- Laederach says that he learned not do one simply to give you a tials and then the MD program.” from Altman’s approach to getting competitive advantage in the other, Former Altman postdoc Alain the most out of the latest compu- because the training for both are Laederach, now a research scientist tational approaches to solve real- long and hard, and any kind of at the Wadsworth Center for Devel- world biological problems. “In superficial reason to do it cannot opmental Genetics and Bioinfor- Russ’s lab, I learned to identify bio- weather how physically and intel- matics, says his favorite memory is logical problems that are lectually difficult it is to finish a rather terrifying experience tractable computationally and iden- them.” And when it comes to look- involving the grant proposal for the tify the best approach,” says Laeder- ing for the right MD/PhD program, Simbios group within Altman’s ach. “I also learned to identify the Altman says that the quality of the NIH center, which was around 250 medical applications of the biology, PhD training should be the decid- pages long. “Three days before it which is critical to obtaining funding factor. “Medical school is basi- was due, Russ decided we needed ing from NIH.”

>NAMING NAMES

Any number of scientific greats have been shaped over the including RiboWeb, a semantic application for Internet- years in Altman’s lab. Here are just a few of those who based, collaborative molecular biology. earned their stripes with him. SEAN MOONEY JEFF CHANG Mooney arrived in Altman’s lab as a postdoc and started According to Altman, Chang arrived in his lab with coding BioE2E, an organization geared toward grad students and skills at a level he has yet to see since. Chang actually postdocs interested in the entrepreneurial side of joined the lab as a sophomore and later proceeded to biotechnology. He is currently an assistant professor write a “trail blazing” thesis on natural language text in the Department of Medical and Molecular Genetics mining, Altman says. He is currently gearing up for a at the Indiana University School of Medicine. postdoc at the Duke Institute for Genome Sciences and Policy. SOUMYA RAYCHAUDHURI During his stint at Altman’s lab, this former MD/PhD RICH CHEN student published nine papers, the record for the most Chen joined the lab as an MD student originally intending papers by any student in the lab. Currently, Raychaudhuri to do a short rotation as a researcher. But, as fate would is a research fellow in the program of medical and popula- have it, a local Silicon Valley venture capitalist happened tion genetics at the Broad Institute. to see a presentation he and fellow labmate Ramon Felciano gave and offered the students several million OLGA TROYANSKAYA dollars to start up the company now known as Ingenuity. Troyanskaya says Altman taught her to be suspicious of her results and how to pick the best collaborators, RAMON FELCIANO among many other lessons. Currently, she is an assistant During his stint with Altman, Felciano co-founded professor at Princeton University’s Lewis-Sigler Institute Ingenuity, where he acts as both chief technology officer for Integrative Genomics, where she works on computa- and vice president of R&D. He has also led the develop- tional techniques for genomic data integration, micro- ment of many informatics and Web-based projects, array analysis, and pathway identificiation.

SEPTEMBER 2008 GENOME TECHNOLOGY 65

Blunt End HUMOR, WE HOPE

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Cambridge Healthtech Institute’s Sixth Annual The only Discovery on October 20-23, 2008 dedicated “Hot World Trade Center • Boston, MA Targets” TARGET Exhibits: October 21-23, 2008 event!

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