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Researchers Advance 454-Based Multiplexing As Roche Preps its Own Sets for Fall Launch

German researchers have developed a barcoding method for 454 Life Sciences’ sequencing platform that allows them to sequence large numbers of random DNA samples in parallel.
The ligation-based approach, which the scientists published last week in Nucleic Acids Research, is not the first barcoding technique for the 454 system. For example, a research team from the University of Copenhagen published a similar method earlier this year in Public Library of Science One (see In Sequence 2/20/2007). In addition, Roche is planning to release two barcoding sets for the 454 platform this fall, which will compete with these and other user-developed approaches.
Unlike other methods, the new approach, developed at the Max Planck Institute for Evolutionary Anthropology in Leipzig, can be applied to all types of random double-stranded DNA, for example shotgun libraries from long-range amplicons or plasmids.
“The work by the German group is very good ... and they have shown how the methodology is working,” said Mostafa Ronaghi, a researcher at the Stanford Genome Technology Center, in an e-mail message. He and Poornima Parameswaran at Stanford have developed, and are planning to publish, their own barcoding method, which is “a good way to get more out of 454 runs.”
After realizing that the 454 platform is not suitable for smaller sequencing projects that require less than one-sixteenth of 454’s picotiter plate — the smallest subdivision available for the platform — the Max Planck researchers, led by Matthias Meyer, a graduate student in Michael Hofreiter’s group, decided to develop their own barcoding method.
As part of that method, they ligate identical double-stranded barcoding adaptors to both ends of the sample DNA and cleave half the adaptors off with a restriction enzyme. The resulting molecules, which carry two identical tags, are ready for standard 454 sample preparation.
The tags are designed to be “particularly robust to sequencing errors associated with homopolymers, a well-known problem in 454 sequencing,” the researchers write in their article.
Unlike other barcoding approaches, which include a tag in sample-specific PCR primers, these barcoding adaptors can be used to tag any sample. This makes the method more flexible because researchers may decide only after the PCR reaction whether they want to clone the PCR product for Sanger sequencing, for example, or tag it for 454 sequencing.
“It’s a very simple and quite efficient method,” Meyer told In Sequence last week. He found that the tagging efficiency is high, giving the researchers no more than two-fold differences in the sequence read representation from different samples. Uneven sequence representation has been a problem, for example, with other barcoding methods that include the tags in the PCR primer.
Using their method, the researchers shotgun-sequenced six human mitochondrial genomes on a small region of the GS 20 picotiter plate.
They are now also using the method to analyze ancient DNA, pooling amplicons from different targets from one individual. Up until now, the “gold standard” for sequencing ancient DNA has been to PCR-clone a DNA segment and sequence multiple clones from one amplicon by Sanger sequencing in order to identify base damage and judge contamination. Now, by sequencing pools of amplicons on the 454 platform, they can omit the cloning step.
In their NAR article, the researchers used six-base tags, allowing them to pool up to 72 samples that differ by at least two bases. Since submitting the article, they have increased the tag length by one base and Meyer said he sees no reason why they could not increase it even further.
To keep the cost for barcoding adaptors low, researchers could combine a strategy of subdividing the picotiter plate and barcoding samples within a subdivision, he said.

The tags are designed to be “particularly robust to sequencing errors associated with homopolymers, a well-known problem in 454 sequencing.”

A single run on the GS FLX would be sufficient to shotgun-sequence 250 mitochondrial genomes, Meyer has calculated. Sequencing these samples by the Sanger method would be more expensive, he claimed, although he did not know by how much.
In addition, the 454 shotgun sequencing method is “at least ten times quicker” and “much simpler” than Sanger-based primer walking, he estimated.
Meyer does not believe that his approach is currently suitable for other next-generation platforms since the tags would eat up too much of the total read length, which is much shorter than the 250-base pair average reads of the GS FLX. But that will almost certainly change in the future, when read lengths for the other platforms increase, he said.
Roche to Roll out Tag Sets
In the meantime, Roche has been developing its own barcodes, which it calls “multiplex identifiers,” or MIDs, using 10-base tags. The company plans to launch two sets of these about three months from now, according to Tim Harkins, the company’s marketing manager for genome sequencing.
“By using 10-mers of specific sequences, we can ensure that primer synthesis errors, PCR errors, and system errors will not collectively lead to inaccurate assignments of sequencing reads to samples,” he told In Sequence by e-mail this week.
The first set of barcodes will be a collection of 12 different tags “that can be used with any type of sample, such as BACs, microbial organisms, or cDNA samples,” according to Harkins.
These will incorporate the 10-base identifier in the adapters that are ligated to the DNA sample during the library preparation. These oligos will contain the 10-base sequence between the “key tag,” which is used for signal calibration and quality control, and the target sequence. Roche plans to release sequence information for the 12 barcodes once beta-testing is complete. The company will also provide software this fall to support the 12 MIDs, as well as customer-generated identifiers.
The second set will comprise 96 “virtual” identifiers, for which the company will make sequence information available, along with support documentation. “Users can make their own MID sets to meet their specific demands,” Harkins said.

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