Microfluidics company Fluidigm has developed a digital PCR-based kit for quantifying second-gen sequencing libraries, and said it believes the tool will save users time and money and enable them to sequence using less starting material.
Fluidigm, which recently postponed its initial public offering (see Short Reads, in this issue), hopes that the new product — its first for next-gen sequencing — will expand the market for two of its platforms. The company is also working on additional sequencing-related applications to run on the instruments.
“We figure this opens up 1,000 new sites for us to market our device to,” Fluidigm President and CEO Gajus Worthington told In Sequence.
The company’s new kit, called SlingShot, was conceived at Stanford University and developed over the last six months or so. Using Fluidigm’s digital PCR arrays, it is designed to accurately quantify the concentration of DNA libraries prior to amplification.
Knowing this concentration precisely is required to determine how much of the library to use in the amplification step — emulsion PCR in the case of the 454 and SOLiD platforms, and cluster amplification in the case of the Illumina Genome Analyzer.
Fluidigm has already released three assay kit for quantifying 454 shotgun and multiplex libraries, as well as Illumina and SOLiD libraries.
Each kit, which contains a single-use digital PCR array and reagents, costs on the order of $350 and can quantify up to six samples in parallel. The chip has 12 panels, each with 765 reaction chambers, in which the PCR reactions take place.
The DNA library is diluted and introduced into the chip so that no more than one DNA molecule enters each chamber. After the PCR reaction, the software counts the number of chambers with PCR products and calculates the concentration of “amplifiable” DNA in the library. According to the company, the assay takes less than four hours.
The assay runs on two of the company’s platforms: the $200,000 BioMark system, which Fluidigm markets for digital PCR applications, TaqMan-based SNP genotyping, and TaqMan-based gene-expression analysis; and the $100,000 EP1 system, a stripped-down version that can perform digital PCR application and TaqMan-based SNP genotyping but lacks the BioMark’s real-time capabilities.
Fluidigm believes that current methods for calibrating sequencing libraries are less accurate than SlingShot, so scientists sometimes need to perform costly and time-consuming titration runs on their sequencers. SlingShot, it says, eliminates this step. “It saves time and money and it also unburdens the [sequencing] machines from doing titrations,” according to Worthington.
Also, some existing calibration methods require large amounts of DNA, he said, whereas SlingShot only takes picograms of DNA. This allows users to sequence from small amounts of DNA, such as those found in clinical or degraded samples.
The SlingShot method was used in a recent paper published in PNAS by the Stanford University group run by Steve Quake, who is a co-founder of Fluidigm. The assay is now used routinely at Stanford, where Richard White, 454 lead operator in Quake’s lab, developed it originally for the 454 sequencing platform.
“We wanted to have a calibration method that would save us cost and allow us to be more high-throughput,” he told In Sequence.
“We figure this opens up 1,000 new sites for us to market our device to.”
White and his colleagues now use the assay to quantify libraries from several different sequencing platforms, including Illumina’s Genome Analyzer, Applied Biosystems’ SOLiD, and — most recently — Helicos BioSciences’ HeliScope, which Stanford obtained last month.
“All the big sequencers are going to have this problem,” White said. “They are going to need a method to absolutely calibrate how many molecules they are loading on a flowcell, or a bead.”
A handful of other labs are using the SlingShot assay, including groups at the National Cancer Institute, the J. Craig Venter Institute, and the University of Maryland.
Researchers at the University of Maryland previously used either Agilent’s BioAnalyzer or fluorometry to quantify their 454 or Illumina sequencing libraries and found both methods to be “somewhat inconsistent,” according to Kristie Jones, a laboratory supervisor at the university’s Institute for Genome Sciences.
“The biggest advantage we’ve seen so far with the Fluidigm SlingShot assay is that we’ve been able to accurately quantify DNA libraries that were not quantifiable at all on the BioAnalyzer, or with fluorometry,” she said. “It turns out there was plenty of DNA to sequence in these libraries, but our previous methods of quantifying them just weren’t able to detect DNA concentrations that low.”
The Wellcome Trust Sanger Institute expects to obtain a Fluidigm instrument this month and to test it over the next few weeks to see whether it has advantages for quantifying Illumina DNA libraries. Though it is more expensive and possibly more time-consuming than its current qPCR-based method, “we are hopeful that it may be more accurate,” according to Harold Swerdlow, head of sequencing technology at the Sanger Institute.
“Any improvement in accurate quantification of cluster numbers pre-amplification would be a boon,” he said, as long as “it leads to a substantive improvement in the actual reproducibility of obtained cluster counts on the Illumina GA instruments.”
According to Matthias Meyer, who published a qPCR-based method for quantifying 454 sequencing libraries about a year ago (see In Sequence 1/8/2008), qPCR works well for about 80 percent of all 454 libraries in his experience, but “there is certainly room for improvement.”
He said the main advantage of digital PCR is that it does not require each library molecule to be amplified with equal efficiency. “On paper, their methodology is appealing and should be an improvement over qPCR, as long as there are no hidden caveats,” he told In Sequence by e-mail.
While qPCR is “pretty cheap,” requiring only standard lab equipment and performing well in “most of the cases,” digital PCR “is likely to be even more accurate and consistent,” he said. However, it requires Fluidigm’s specialized instrument and consumables, he noted.
According to Worthington, Fluidigm believes the new sequencing applications will help it market its systems to additional users. “Historically, we have been marketing the BioMark system to people doing gene expression and genotyping, and there is a little bit of overlap now between those users and the next-gen sequencing group, mainly in the area of whole-transcriptome analysis,” he said. “But there isn’t a lot of overlap [with] the high-throughput sequencing groups.”
The company, which says it is currently the only firm with a digital PCR system on the market, is working on other applications for second-gen sequencing as well. “There are other parts of the preparation for high-throughput sequencing that are cumbersome or difficult,” according to Worthington.
One of them is barcoding samples, so sequencing runs can be multiplexed. Fluidigm hopes to launch a product for that application during the first half of next year. “We are working on chips that would make that really easy,” he said.
“The way we think about sequencing is, we are developing library quantitation and other preparative devices that complement the high-throughput sequencers.”