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qPCR Method That Improves 454 Sample Prep May Also Help Illumina, SOLiD Users

Scientists in Germany have found a new way to quantify 454 sequencing libraries, allowing them to start with less DNA and eliminating the need for costly and labor-intensive 454 titration sequencing runs.
The new method, which the researchers published in a recent issue of Nucleic Acids Research, might also be used on Illumina’s Genome Analyzer and Applied Biosystems’ SOLiD system.
It promises to benefit any researcher who has limited amounts of DNA available for sequencing.
To make a 454 sequencing library, researchers usually start with several micrograms of sample DNA, according to Matthias Meyer, a graduate student at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, and the lead author of the article. The reason for that is not that so much DNA is required for 454 sequencing, but that the sequencing library must have enough DNA left to be quantified accurately. That is necessary, he said, to determine how much of the library should go into the emulsion PCR, which amplifies the DNA for sequencing.
But the Max Planck scientists, working with DNA extracted from Neandertal bones, only have little starting material available. “In the best case, it’s in the nanogram [range] but usually it’s in the picogram range,” Meyer told In Sequence last week.
After preparing the 454 libraries, which involves attaching two different adapters and isolating DNA that carries both adapters, not enough DNA was left to be quantified using the methods recommended by Roche, either Agilent’s BioAnalyzer or Ribogreen quantification.
Not knowing how much DNA was in their libraries, “what we had to do all the time was to blindly run these libraries on the 454 machine to empirically find a concentration that actually works for sequencing,” Meyer said.
The researchers instead decided to use quantitative PCR to quantify the sequencing libraries, a more sensitive method. “This is really ridiculously simple, actually. But it immediately gave good sequencing results, [because] we could precisely quantify libraries that could not be quantified before,” he said.
The new qPCR quantification method means the scientists are no longer wasting precious sequencing runs, or titration runs, just to find the right concentration of DNA to use in the emulsion PCR. “Before, we were spending three, four, five [sequencing] lanes in order to find a lane that has relatively good sequence numbers, and now we are consistently able to get a good number of sequence [reads] from each lane we are sequencing,” he said. Each 16-lane run cost around $10,000 in reagents.
With their new protocol, the scientists are now able to start with as little as 50 picograms of starting DNA with no titration runs, a potential benefit for any researcher with limited amounts of biological material available for sequencing.
Several researchers have experienced this problem. “It will be greatly helpful in 454 sequencing,” Yuan Gao, an assistant professor at Virginia Commonwealth University, commented by e-mail. “Most importantly, the starting material could be far less.”
Gao has experience with 454 and Illumina sequencers. “In terms of lowering the cost [of 454 sequencing], it will save at least $4,000 due to the elimination of the titration run before the real run,” he estimated, in addition to labor and time savings.
Others are less sure the method will help with their sequencing projects. “We have worked with vanishing low amounts of DNA, and it is a real pain, but the problem is not so much quantitation as it is getting PCR products that are not contaminated with background DNA samples from reagents,” said Mitchell Sogin, director of the Josephine Bay Paul Center at the Marine Biological Laboratory in Woods Hole, in an e-mail message. “The technology described here might be very helpful for genomic studies and cDNA studies, but I am not sure [it] will solve our low biomass problems.”

“This is really ridiculously simple, actually. But it immediately gave good sequencing results.”

In addition to sequencing ancient DNA samples, the new method has helped Meyer and his colleagues develop new 454 library preparation protocols, for example for RNA sequencing. “Before, you needed micrograms of RNA, and that’s really difficult, because for many precious samples, it’s hard to get so much material,” Meyer said. Now, “even if you get very low quantities out of your protocol, you can still get an estimate of how much you’ve got and what percentage you have recovered from your starting material.”
But Meyer says his team is using the qPCR quantification method, which adds about 2 to 3 hours to the sample prep process, not only to quantify libraries containing little DNA but also to analyze highly concentrated sequencing libraries, because “it appears to us that it’s slightly more precise” than the quantification methods recommended by Roche. As a result, they no longer need to perform expensive titration runs, which Roche recommends, even when starting with micrograms of DNA.
The method could also work with Illumina’s Genome Analyzer and ABI’s SOLiD sequencer, Meyer suggested, which, like 454, use two adapters. The only requirement, he said, is that the sequences of these adapters be known. However, he does not know of anyone who has tested the method with these systems.
Both Illumina’s and ABI’s platforms require nanograms to micrograms of starting DNA, although only a fraction of that original DNA, after amplification, eventually goes into the sequencer. “I’m sure they have the very same limitation, that once you have turned your sample into a library, you have too little of the library, [and] you can’t determine the concentration anymore,” Meyer said.
According to Gao, the method could indeed be useful for Illumina’s system, although this platform does not require titration runs. However, “the starting material will be lowered, [the] concentration determination will be more accurate, and the number of clusters desired can be controlled better,” Gao told In Sequence by e-mail. “I will definitely give it a try.”
“Getting enough starting material is often a problem, and will even require an initial amplification method such as whole genome amplification or degenerate oligonucleotide-based amplification,” said Anoja Perera, lab manager for molecular biology facility at the Stowers Institute for Medical Research in Kansas City, by e-mail. Her lab is currently using Illumina’s protocols but plans to work on making these more efficient and adapting them to the institute’s needs. “We will surely look at papers like [this one] for guidance.” 
The method could be further optimized, Meyer said, and should be tested on larger sets of samples to find its limitations. But he and his colleagues are not planning to do that “because it works well for us; we are quite happy now.”
It is unclear whether Roche will implement the method in its library protocols for the 454 sequencer anytime soon. “The example in the NAR publication is a nice protocol to reduce the amount of starting material,” a Roche spokesperson told In Sequence by e-mail. The company is working on several projects that couple the use of qPCR with the GS FLX, he said. However, “it is still too early to comment on when we may see … protocols that couple the two platforms.”

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