November 16, 2010 | Last year, shortly after completing work on rock music legend Ozzy Osbourne’s memoir, I Am Ozzy, The Sunday Times (London) reporter Chris Ayres was sitting next to Knome CEO Jorge Conde at the TedMed conference in San Diego. “When Ozzy and I began to do the weekly ‘Dr Ozzy’ column for The Sunday Times—now also in Rolling Stone—I got the idea to ask [Knome] about possibly sequencing Ozzy’s genome as a one-off article. It snowballed from there,” says Ayres.
John Michael “Ozzy” Osbourne, the former lead singer of Black Sabbath, has become the latest member of the celebrity genome club, joining Glenn Close, Archbishop Desmond Tutu, Jim Watson, Craig Venter, Henry ‘Skip’ Gates and others. On October 24, Osbourne penned an absorbing 3,500-word account in the Sunday Times Magazine of his reaction to being presented with his personal genome on a souped-up thumb drive.
Osbourne is a curious character for a genome sequencing project, but his lively musical career, characterized by controversial episodes involving the occasional decapitation of birds and bats onstage and the consumption of copious quantities of drugs and alcohol, makes Ayres and others curious as to whether the secret of his extraordinary longevity lies in the sequence of his DNA.
Osbourne, who described himself as “a rock star, not Brain of Britain,” initially needed some convincing to go along. “The only ‘genome’ I’d ever heard of was the kind you find at the bottom of the garden with a little white beard and a pointy red hat,” he wrote. “The only Gene I know anything about is the one in Kiss.” But he came around. After all, he wrote, “Given the swimming pools of booze I’ve guzzled over the years—not to mention all the [drugs]… there’s really no plausible medical reason why I should still be alive. Maybe my DNA could say why.”
Supporting Osbourne was a tight three-piece outfit. The sequencing was conducted at St Louis-based Cofactor Genomics, performed on the Life Technologies SOLiD 4 platform, which also assisted in the early bioinformatic analysis. The final genomic interpretation and presentation was handled by Knome.
Cofactor Genomics was established in 2008 by former members of The Genome Center (TGC) at Washington University, St Louis, who worked in the technology development group under Elaine Mardis. “We’d see people come to The Genome Center with cool projects, but there were reasons why they couldn’t take it on—capacity, funding issues, etc.,” says Jon Armstrong, Cofactor’s chief marketing officer.
Armstrong and colleagues had plenty of experience installing new sequencing platforms, including the very first Solexa machine (serial number #001). “Because of Elaine Mardis and Rick Wilson, we couldn’t have done what we’ve done without being under their tutelage. We had a unique and privileged outlook on the emergence of next-gen sequencing and how that hardware interfaces with genomics. We’ve been involved in the development of those protocols and producing very high quality data under time constraints and doing it right the first time.”
Cofactor offers Life Technologies’ SOLiD 4 and Illumina’s GA IIx instruments, and is contemplating whether to acquire Illumina’s HiSeq 2000, but needs “to understand what that machine means on our operational and fiscal side,” says Armstrong. Other platforms, such as Ion Torrent, might prove a better fit depending on how the sales pipeline evolves. Sometimes, “using a paring knife instead of a cleaver is a better choice,” he says. Armstrong says Cofactor has “a very strong analysis component,” and a good relationship with NGS software company NovoCraft (Malaysia), using NovoAlign for Illumina work and NovoAlign CS for some of the SOLiD data.
Early work focuses mostly on RNA sequencing, with steady business coming from the biofuels and agricultural sector. Cofactor is also preparing a major project with Sigma to sequence the genomes of several strains of rat and build a publicly available database of rat genomic variation.
As for Ozzy, Armstrong was approached by Life Technologies asking if he would be interested in “an amazing opportunity” that had come up. He was told that Life Technologies “needed this done right the first time.” Ozzy was not Cofactor’s first whole genome sequence, but it was Cofactor’s first serious use of the SOLiD 4 machine. Cofactor generated 39 Gigabases of Osbourne’s sequence (13X coverage) over about three weeks using both long-insert mate pairs and fragment reads. (Human genomes are usually sequenced to 30X coverage, but Osbourne’s sequence was time sensitive.) About 70 percent of the DNA reads were mappable, which he calls “very good.”
Houston-based Matt Dyer has a field-based position with Life Technologies, supporting Midwest users of the SOLiD platform on issues including experimental design and data analysis. “Jarret [Glasscock, Cofactor CEO] contacted me, and said they needed some help for an urgent, once-in-a-lifetime project,” says Dyer, who didn’t need to be asked twice. “I’m a big fan of biology and bioinformatics—though not Black Sabbath necessarily!”
Because of the urgency of the project, Dyer used cloud resources offered by Penguin Computing (Penguin On Demand). After Cofactor uploaded the sequence data to the cloud, Dyer logged on and took over, with immediate access to thousands of compute nodes. No read filtering was performed. “We don’t do any filtering—we let the mapping do the filtering,” says Dyer. “If the read is bad, it won’t map to the reference.”
BioScope is Life Technologies’ integrated framework that allows users to perform secondary analysis (mapping sequence back to the reference) and tertiary analysis (e.g. SNP calling, indels, CNVs), all within a single software package. BioScope can run on many different types of hardware, but Dyer says, “for folks who can’t maintain an expensive IT infrastructure, they can use this fee-for-service cloud, buying the CPU hours they need.”
Dyer used BioScope to map Ozzy’s sequence reads to the human reference genome (HG18)—the process took about 8-10 hours. From there, he created BAM files, which were then used for the tertiary analysis. “We gave those pipelines the BAM file and asked to return the SNPs and small indels,” he says. Knome was able to download the SNP and indel data via FTP, while Dyer shipped the full sequence data on a hard drive.
Not gnome, Knome!
“Ozzy is a pretty unique guy with an extraordinary life but he’s still around to talk about it. We thought we could highlight someone, an everyman if you will,” recalls Jorge Conde. He received some external funding (he did not identify the source) while Life Technologies funded the reagent sequencing.
Knome took the assembled sequence and ran it through its internal analysis pipeline, producing a richly annotated genome and a set of interesting DNA variants to select for further analysis. Knome’s director of research, Nathan Pearson, then flew to the UK recently to deliver Osbourne’s results in person, but not before he had conferred with Osbourne’s wife, Sharon, a judge on America’s Got Talent.
“We thought about what it is that makes Ozzy unique,” says Conde. “Given he’s a musician and he’s been diagnosed with a parkinson’s-like tremor, we looked for things associated with nerve function. We found a couple of interesting things—but as Nathan says, ‘We found smoke but no fire.’ There was no ‘Ozzy variant’—that goes without saying.” The interesting variants lie in the genes TTN (associated with deafness and Parkinsonism) and CLTCL1 (brain chemistry). They might merit further study, Conde suggests.
Knome also found a novel variant in the ADH4 alcohol metabolizing gene, which might explain Osbourne’s legendary high tolerance for alcohol. Ironically, Osbourne is highly sensitive to caffeine, and has the genetic variant to prove it. Another interesting variant was in the COMT gene. Osbourne carries two versions of the gene that have been associated with “worrier” and “warrior” behavioral tendencies.
Another interesting tidbit was the ability to screen Osbourne’s genome for traces of Neanderthal DNA, following new evidence of ancient inter-breeding between humans and Neanderthals emerging from the Neanderthal genome study. “We were looking for matches for long segments unique to Neanderthals, and found a couple of pretty long segments [in Osbourne’s genome],” says Conde. “But George Church [Knome co-founder] has 3x as much matching segment.”
Overall, Conde says Knome enjoyed working on Ozzy’s genome. “I don’t think doing celebrity genomes is a business model,” he says, “but we’re thrilled to work on interesting projects as they come along. This was a lot of fun!”
Life Technologies’ Matt Dyer agrees. He says the project was “a great example of how genomics and bioinformatics helps us understand what makes you you and me me.”
As for those front-row Ozzy Osbourne World Tour tickets—the Prince of Darkness descends on St Louis on December 10 and Houston on January 18, 2011—Dyer says: “Still waiting for those!” •