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Stanford, UC Santa Cruz Researchers Develop Digital Array Technique for NGS Counting Applications

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A group of researchers from Stanford University and the University of California, Santa Cruz have developed a technique that combines aspects of microarray technology with next-generation sequencing.

Described in a study published this month in PLoS One, the sequencing bead array, or SBA, technique is "a suspension array for next-gen sequencing platforms" that replaces the optical readout with a label-free digital readout, or barcode, lead author Michael Akhras, a postdoctoral researcher at Stanford, told In Sequence. Rather than using the next-gen sequencing system to evaluate each base pair, next-gen sequencing is used to check for the presence of a target.

"It's a counting assay," Akhras added. "Instead of reading the target region, it is just checking if a target is there or not. And it's digital because we're counting if the target is present and the amount. … We get an exact count of the particular barcode."

In the PLoS One study, the team used Life Technologies' Ion Torrent PGM for the sequencing, which Akhras said was most amenable to the technique because of the PGM's use of beads. It would also be possible to use the technique in conjunction with Roche's 454 GS FLX or GS Junior instruments, and he said he is working on modifying the protocol to work with Illumina's systems.

The team also developed its own analysis software, Sphix.

In the study the researchers wanted to test the assay's ability to genotype human papilloma virus, so they created a synthetic oligonucleotide library of targets that corresponded to 10 different HPV subtypes.

The synthetic oligonucleotide consists of the target hybridization site as well as a barcode unique to that target, and a general priming site. Multiple synthetic oligos are bound to biotinylated beads to create a reporter, each of which is assigned a unique ID that corresponds to a specific target of interest.

Akhras said that in a commercial version, customers would simply purchase the pre-made reporter library. The customer would then combine the reporter library with the PCR products following DNA amplification.

The oligonucleotides bind to their respective targets on the DNA, which activates the reporter. Those reporters are then magnetically selected with streptavidin-coated beads. If one of the targeted regions is not present in the DNA, the reporters with oligos corresponding to that region are washed away. Sequencing primer is added to saturate all the beads, which are then run on, in this case, the PGM. Sequencing identifies the presence or absence of each of the barcodes to determine which DNA targets are present and which are not.

In the study the team tested the technique on its ability to genotype 10 high-risk HPV genotypes associated with cervical cancer progression.

For each genotype, they created reporter libraries from six different emulsion PCR reactions to test reproducibility and sequenced each library individually to test variability.

In the 10-plex reaction, the researchers found slightly less than ideal composition of reporters. While each reporter should ideally make up 10 percent of all the reporters, actual distribution ranged between 6.63 percent and 12.48 percent for each reporter, which the authors attributed to problems inherent with emPCR.

Nevertheless, the researchers were able to identify each of the 10 HPV genotypes with the method, which they confirmed with pyrosequencing on Roche's 454 GS FLX.

Additionally, by testing increasingly diluted samples of target, they established a lower detection limit for the assay at 10 femtomolars, which they said could be lowered further by improved loading onto the PGM chip.

Next, they pooled PCR products from five different HPV genotypes to simulate co-infection and verified that the assay could detect all five.

Once the researchers established that the assay could detect the 10 HPV genotypes, they tested it on clinical samples from 20 cervical tumors. HPV was confirmed in 19 cases, and the assay was concordant with 454 sequencing with the exception of two samples.

Both of those samples represented co-infections of two HPV genotypes. The SBA assay detected both, but sequencing on the GS FLX only detected the dominant genotype, "due to the higher sensitivity and dynamic range for the SBA assay," the authors wrote.

In one sample, neither SBA nor amplicon sequencing on the GS FLX were able to detect an HPV genotype. The researchers created 10 PCR reactions representing each possible HPV genotype, but the failure of either method to detect HPV led the team to conclude that the sample was negative for HPV. The authors wrote that the sample could have been negative, despite its tumor origin, "due to the presence of an uninterrogated HPV genotype," because the DNA extract did not capture any HPV, or the sample could in fact be a "true negative that does not carry anymore HPV virus due to prior cleared infections."

Going forward, Akhras said that the team plans to continue to make improvements to the method. For instance, he said he would like to make it more quantitative. Currently, the assay gives a "yes or no" answer, he said. Making it more quantitative would require different methods for designing the beads, he said. Additionally, he is interested in testing the method on the Ion Proton. The smaller beads of the Proton compared to the PGM should help increase the assay's sensitivity, he said.

Additionally, Akhras said that because the assay needs to read only about 10 base pairs, enough to identify the barcode, the more simple the sequencing system the lower the cost and the faster the turnaround time will be.

Currently, turnaround time to run the assay after the PCR product is generated is about one and a half hours, he said, a time that he is also looking to reduce. He sees the primary application being in rapid pathogen identification, and eventually, if the assay is improved, it could be used in other counting applications like prenatal diagnostics.

He sees it competing primarily with flow cytometry assays like those developed by Luminex or NanoString. "NanoString is very sensitive," he said. "So the question is, 'can we get [SBA] that sensitive?'"

Nevertheless, he said it would have an advantage as next-gen sequencing becomes increasingly adopted in the clinic. Next-gen sequencing machines "are going to be more and more present in the clinic," he said, so the advantage of SBA is that it will be "working with a system that exists" and customers won't have to buy an instrument for a specific assay.

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