In an effort to capture a section of the growing high-throughput sequencing market, Invitrogen last week launched two sample-prep reagent kits for long-range PCR and for PCR product purification and normalization.
The kits, which Invitrogen is marketing for use with next-generation sequencing platforms, are the first of several products that the reagents giant is developing for use with downstream high-throughput sequencing. Banking on its experience with molecular biology enzymes and reagents, the company is hoping to fill a void in the sample prep area left by the sequencing platform vendors.
“I think the instrument providers did a good job at getting these instruments out in the market, but to actually follow it up with good sets of kits and QC’d reagents and pieces that work together, I think it’s still a little bit lacking,” Peter Welch, Invitrogen’s director of R&D for gene regulation, told In Sequence last week.
While second-generation sequencing vendors provide reagents for amplifying DNA prior to the instrument run — be it emulsion PCR or cluster amplification — DNA library preparation workflows are generally long and involve many steps, such as gel purifications and ligations, Welch said.
Moreover, vendors often provide customers with protocols that require them to purchase reagents from third parties, including Invitrogen. “So, putting all that together in an optimized kit, I think, is a natural next step,” he said.
“When you think about what Invitrogen is known for as far as enzymology and PCR [goes], it’s a natural fit for us,” he added. “This is where our core competencies lie.”
Less than a year ago, Invitrogen set out to plant roots in this niche by putting together a new R&D group devoted to developing products for use with high-throughput sequencing systems.
The first fruit of this endeavor is the two new SequalPrep kits, both of which Invitrogen launched last week. The SequalPrep Long PCR kit, which costs $799, contains reagents for long-range PCR reactions that allow users to generate PCR products on the order of 5 to 20 kilobases “pretty much without optimization,” according to Welch. The kit contains two enhancers, each containing unspecified proteins and buffer formulations that customers can use in different concentrations to successfully amplify their DNA.
The Sequal Prep Normalization Plate kit, on the other hand, costs $949 and purifies up to 96 PCR amplicons on a 96-well plate and normalizes their amount. Users add at least 250 nanograms of PCR product to the plate and elute up to 25 nanograms per well, obtaining the same amount of DNA each time within a two- to three-fold concentration range.
Invitrogen hopes that the two kits will be used to amplify regions of the human genome in large numbers of samples to prepare them for high-throughput sequencing, for example for medical resequencing studies.
The first customers the company is targeting are large genome centers with multiple second-generation sequencers, according to Welch. “They are the ones that typically are handling lots of patient or test samples [and] that are currently adding barcodes to each different patient and then pooling [them],” he said.
However, the kits could also be useful for labs with a single second-generation sequencer, and “we even see some benefits of this for people still doing traditional Sanger sequencing,” he said.
Long PCR vs. Gene Capture
The PCR kits will likely also compete with array- or oligonucleotide-based enrichment methods that academic groups and companies have recently been developing, using tools from Roche NimbleGen and Agilent Technologies, for example (see In Sequence 4/8/2008).
But according to Welch, small numbers of genomic regions could still be more easily and cost-effectively amplified by PCR, whereas the new capture methods would be more suitable for amplifying many regions, such as all exons in a sample.
“When you think about what Invitrogen is known for as far as enzymology and PCR [goes], it’s a natural fit for us.”
“It’s really limited by where that tipping point for your lab would be to go from PCR to some other kind of exon enrichment,” he said.
Before launching the kits, Invitrogen tested them both in-house and in collaboration with “several of the major genome institutes,” according to Welch.
One of them is the genome research center at Cold Spring Harbor Laboratory, where Michael Regulski, a research investigator in Dick McCombie’s group, tested the long-range PCR kit on three regions, sized 50, 140, and 550 kilobases.
“This Invitrogen system works very well,” he told In Sequence last week, noting that it performed well for the two smaller regions.
For example, the PCR reaction kit helped him reduce from 20 to 6 the overall number of PCR products needed to cover the 50-kilobase region. “When you target genomic regions, you want to have as long products as possible, because then you have fewer reactions to run,” he explained.
Regulski was also able to cover the 140-kilobase region almost entirely using the long PCR kit, except for a tricky 5-kilobase region that he is still trying to amplify successfully. However, for the 550-kilobase area, it “was much more difficult to get equally good results across the whole region” using the kit, he said.
He also tried the GeneAmp XL PCR kit from Applied Biosystems, which contains rTth DNA polymerase XL, an enzyme blend designed to PCR-amplify DNA in the range of 5 kilobases to 40 kilobases. That kit “works very well, too” he said, and gave better results for the long region than Invitrogen’s product. “I don’t think there is a golden bullet,” he said.
The Cold Spring Harbor scientists are still using Invitrogen’s kit for “a couple of systems that give a nice, clean product,” according to Regulski. A PCR-based approach is still the method of choice today for sequencing “a shorter region in multiple patients,” he added.
However, for larger regions, the new capture methods are better suited, he said. “When you get to regions that are 500 kilobases, I would think capturing genomic fragments on a chip is probably a better way to go, and certainly [requires] much less labor,” he said.
“I think eventually, the hybridization-based methods for targeting genomic regions will probably overtake the field,” he said. “In the end, they will be cheap enough.”
In fact, Invitrogen is working on its own enrichment method as an alternative to PCR. “We are definitely interested in other ways of enriching; we see a lot of value there,” Welch said. “Certainly the arrays are getting a lot of press right now, but there are lots of ways to solve that problem.”
Besides methods for genomic enrichment, the company is “very interested” in sample prep methods relating to epigenetic studies, according to Welch, such as chromatin immunoprecipitation and DNA methylation, which are increasingly coupled with second-generation sequencers.
The company also wants to develop new products for small RNA discovery and profiling. It already sells miRNA microarrays and qRT-PCR kits under its NCode brand but “we like to expand that and keep that product line growing and current,” Welch said.
Invitrogen has not decided whether it will sell upcoming kits for use with second-generation sequencing platforms solely on its own or whether it might team up with the platform vendors.
“We certainly sell reagents to each of the providers already today and we buy reagents from them for some of our things, so there is already a lot of cross-fertilization,” Welch said. “Whether or not this will expand further into the next-gen [sequencing] market is still to be determined.”