By Monica Heger
Last week, NuGen launched a sample prep kit enabling multiplexing of up to 384 samples per sequencing lane for next-gen sequencing on Illumina platforms. The Encore 384 Multiplex System uses the same workflow as its other sample prep systems but has been automated on Caliper's Sciclone NGS Workstation.
NuGen said the new product is an example of its direction for next-gen sequencing sample prep: flexible and automated.
"We're striving to bring the sample prep step of next-gen sequencing up to the level of the throughput and capacity of what the sequencing platforms are offering, so it's not a bottleneck," Steven Kain, NuGen's director of product marketing, told In Sequence.
Illumina itself offers multiplexing on its machines of up to 12 samples per lane (96 samples per flow cell). Its sample-prep kits contain 12 unique oligonucleotides, each six bases long, to tag libraries.
NuGen's previous sample-prep kits enabled multiplexing of only up to 16 samples per lane. The key to achieving the jump to 384 samples, or 3,072 samples per flow cell, was by increasing the length of the barcodes from four bases to eight bases.
"The main thing we had to do is increase the information content in the barcodes," said Joe Don Heath, NuGen's vice president of global technical services and automation. Increasing the length of the barcode to eight bases allowed them to contain enough information to "unambiguously separate one library from the other," he added.
The choice of the sequences within the barcode is also important, Kain added. The barcodes have an equal representation of bases at each position, which is important for the Illumina platforms. "You have to have an equal representation in order to get an efficient cluster call on the sequencing step," Kain said.
In addition, the barcodes have to not only unambiguously separate the different libraries, but they should also be "forgiving of sequencing errors," Kain said. So, the barcodes were also designed to tolerate changes of up to three bases without being replicated in another barcode.
The kit could be used for several applications, including as a way to screen a large number of samples with low-pass sequencing, to sequence deeply a small region of interest, or for amplicon sequencing, said Kain.
For instance, said Kain, it could be used to "track how viral sequences differ across a range of individual cell types infected with the virus." Alternatively, "maybe there's a defined region within a transcriptome or genome that's indicative of a certain drug response or a certain disease state," he added. Many samples could be multiplexed and screened for those markers, and then followed up with deeper or broader sequencing.
Kain envisioned that academic researchers, large genome centers, and biotech and pharmaceutical companies that have adopted sequencing at a large scale would be the most likely customers for the new kit. He said the company has also received interest from its multinational customers who would use the barcodes as a way of tracking samples.
While the initial version is specific to the Illumina platform, Kain said that it could be adapted to other sequencing platforms simply by using the appropriate sequencing primers, which the company plans to do this year "for other leading platforms."
The company's main competitors are the sequencing vendors themselves such as Illumina, Life Technologies, Roche 454, and Ion Torrent, which offer reagent systems for library construction on their respective platforms. Illumina's multiplexing kit, which can pool 12 samples in each lane, was the highest level of multiplexing available aside from NuGen products.
Looking ahead, Heath said that NuGen will be focused on automation. "As sequencing throughput increases and the demand for more sequencing keeps growing, genome centers and larger pharmaceutical and biotech companies are demanding assays that can be easily automated," he said.
One way in which the company has focused on automation is through its partnerships. In February, NuGen and Caliper Life Sciences announced they would collaborate to co-develop automated library construction for next-gen sequencing on Caliper's Sciclone system (IS 2/1/2011), which now includes the Encore 384 kit.
And in April, NuGen said it would work with IntegenX to automate its sample prep workflows on IntegenX's Apollo series systems (IS 4/12/2011).
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