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Large Genome Centers Replace Sanger With Second-Gen Sequencers for Many Applications

Fewer than four years after 454 Life Sciences became the first second-generation sequencing company to launch its platform, large genome centers that had been heavily invested in Sanger sequencing technology have replaced many of their high-throughput capillary electrophoresis instruments — most of them Applied Biosystems 3730s — with a mix of new tools from 454, Illumina, and Applied Biosystems.
The new technologies, which differ in read length, throughput, cost, and other metrics, have taken over many applications from Sanger sequencing and have opened the door to projects that were impractical or too costly before.
However, Sanger sequencing remains the technology of choice for certain applications, such as clone-based sequencing, and is unlikely to disappear any time soon, according to several of the centers.
Data from seven non-commercial genome centers (see table below) — the Broad Institute of MIT and Harvard, Baylor College of Medicine’s Human Genome Sequencing Center, the Genome Center at Washington University St. Louis, the Wellcome Trust Sanger Institute, Beijing Genomics Institute Shenzhen, the J. Craig Venter Institute, and the Joint Genome Institute — show that in total, the number of second-generation sequencers operated by the centers — 149 — has still not surpassed the number of ABI 3730s, though their data throughput is orders of magnitude greater.
All seven genome centers were involved in a variety of genome-sequencing projects and had been heavily invested in Sanger technology. Most, if not all, have recently retired part of their Sanger base (see In Sequence 3/25/2008).
Collectively, the centers currently operate 86 Illumina Genome Analyzers, 41 Genome Sequencer FLXs from Roche’s 454, and 21 ABI SOLiD systems. However, individually they have opted for different technology mixes.
For example, while the Sanger Institute maintains five ABI SOLiDs, two 454 GS FLXs, and 26 Illumina GAs — probably the largest number of Illumina sequencers of any genome center — Baylor’s HGSC runs 10 GS FLXs, six ABI SOLiDs, and two Illumina GAs.
Cutting Capillary Systems
The Broad Institute has reduced its fleet of ABI 3730s by about 60 percent over the last year; it had more than 100 of the high-throughput capillary sequencers a year ago (see In Sequence 9/4/2007), a number that has shrunk to 39 these days. It replaced them with a mix of 20 Illumina Genome Analyzers, 10 454 Genome Sequencers FLXs, and three ABI SOLiD systems.
“It’s all three [platforms], really, that have taken up the work that we have moved off the 3730,” Chad Nusbaum, co-director of the genome biology program at the Broad, told In Sequence last week. “More than anything, it’s been driven by the cost of 3730 data being so much higher than all the others.”

“3730s will live on, even in large genome centers.”

Likewise, the Genome Center at Washington University has retired about two-thirds of its 135 3730s over the last year or so, replacing them with 14 Illumina GAs, five 454 GS FLXs — to be increased to eight — and three ABI SOLiDs.
“The reduction is due to the increasing use of next-gen sequencers of all types,” according to Elaine Mardis, co-director of the center. “Depending on the application, we may choose any of these platforms.”
Since it began buying second-gen systems, the UK’s Wellcome Trust Sanger Institute has reduced the number of 3730s almost by half, from 75 to 40.

“Most of our capillary machine reduction has been due to projects moving to the Illumina platform,” said Carol Churcher, head of sequencing operations at the Sanger Institute.
“Each center varies their usage and types of projects where they need different technology solutions,” said Eric Vennemeyer, senior product line manager of genetic analysis systems at Applied Biosystems. “Many customers are recognizing the complementary nature of next gen and capillary electrophoresis systems.”
Superseding Sanger
454 Life Sciences has vowed that its platform will eventually replace Sanger sequencing, and CEO Chris McLeod stated last week that with the firm’s new Titanium reagents and software, which enable an average read length of 350 to 400 base pairs and a Q20 length to 400 base pairs, 454 has been able to “now displace Sanger sequencing for a lot of sequencing applications.” (See In Sequence 9/30/2008)
Indeed, several sequencing centers said that 454’s longer reads have taken over some, but not all remaining, Sanger applications.
“It is getting close to true, but really only for the shotgun phase” of a genome-sequencing project, said Bruce Roe of the Advanced Center for Genome Technology at the University of Oklahoma. “We still need Sanger sequencing on the ABI 3730s if we want to close any remaining gaps and clear up ambiguous regions.”
Also, until now, he said, 454’s Newbler assembler cut out repeat sequences from the genome. However, the longer Titanium reads, in combination with an update of the Newbler software, should take care of that problem.
Roe, a professor in the department of chemistry and biochemistry at the University of Oklahoma, is one of the earliest 454 customers, and his group has developed several protocols to improve the platform’s performance and to lower the cost for users (see In Sequence’s sister publication, GenomeWeb Daily News 9/5/2006).
“I love the idea that 454 would displace Sanger, but it’s not complete yet,” said Nusbaum. “They certainly substantially reduced our dependency on Sanger.”
Nusbaum said that while 454’s reads are now “nearly as accurate” as Sanger reads, the quality of paired reads “is far superior still on Sanger because you get full-length reads at any distance you can make a plasmid for.”
Also, he said, none of the new technologies enables researchers to retrieve the sequenced clone for further study. “If you want to sequence a cDNA library and want to be able to go back to that cDNA clone, you pretty much have to do it on a capillary,” he said.
Finally, Sanger technology is still the best way to sequence “a few samples of this and a few samples of that,” he said, even accounting for the possibility of tagging samples with barcodes and pooling them for a single 454 run. “If you only need 10 or 100 reads, the cost of a [454] library is already more than the cost of a run from the capillary technology,” he said.
For these reasons, the Broad Institute is hanging on to its remaining 3730s for now. “We still run them, they are expensive to run, … but if you want to sequence BAC ends, you have to do it that way,” he said, adding “that’s all stuff we want to make go away, of course.”
“We agree that the genome centers and other laboratories will be using 3730 units for the foreseeable future,” according to Vennemeyer.
The Sanger Institute has not yet had access to the 454 Titanium upgrade, and is currently using 454’s technology for SNP discovery. It is also using or assessing a mix of 454 and other technologies for pathogen sequencing and comparative sequencing projects.
Sanger sequencing is still more suitable than other platforms for small projects, such as viral sequencing, construct verification, and genome finishing, according to Churcher.
Harold Swerdlow, head of sequencing technology at the Sanger, said “454 has not yet had a serious impact on the number of capillary machines” at the institute, which it still uses for large de novo whole-genome shotgun projects. However, he said the Sanger is no longer taking on these types of projects.
While the institute has moved some Sanger applications to Illumina’s platform, “most of our Illumina capacity is currently being used for exploring new applications that were not practical to do on capillaries,” such as resequencing multiple cancer genome or the 1000 Genomes Project,” said Swerdlow. “So it is more the case of doing novel projects on the Illumina platform, rather than shifting projects away from capillary instruments.”
Mardis said that Wash U has begun to replace Sanger with 454 but pointed out that Sanger sequencing is still needed for certain applications, such as the custom primer reactions used in sequence finishing. Also, “clone-based sequencing is typically the only way to sort through complex structural regions such as the MHC and HLA regions,” she said. Finally, lower-end users likely have no use for the large data quantity generated in a single 454 run, she said.
Wash U’s Genome Center still uses its Sanger fleet for a number of projects that are funded by the National Human Genome Research Institute, including a human structural variation project that requires fosmid end-sequencing and finishing selected fosmids (see In Sequence 5/15/2007).
“So, 3730s will live on, even in large genome centers,” Mardis said.
In the meantime, new genome centers, including Cold Spring Harbor Laboratory, the National Center for Genome Resources, and the Ontario Institute for Cancer Research, do not carry the Sanger legacy and have instead decided to bet almost exclusively on second-generation sequencing technologies.

Sequencing Platforms at Selected Genome Centers
ABI 3730
454 GS FLX
Illumina GA
Danaher/Church Polonator
Broad Institute of MIT and Harvard
Baylor College of Medicine, Human Genome Sequencing Center
Washington University St. Louis, Genome Center
(8 planned)
Wellcome Trust Sanger Institute
Beijing Genomics Institute
(also 107 MegaBace 1,000)
J. Craig Venter Institute
ca. 20
Joint Genome Institute
(7 by end
of Oct.)
ca. 267

Source: Presentations at CHI’s Exploring Next-Generation Sequencing conference, Sept. 2008, and interviews.

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