ORLANDO, FLORIDA – PCR technology startup N6 Tec has launched a new plate-based thermocycling platform where the temperature of each well can be controlled separately for next-generation sequencing library preparation.
Named IconPCR, the instrument promises to enable sample quantification, amplification, and normalization in one workflow, delivering a desired DNA output for each well.
"We call this IconPCR, which stands for individually controlled PCR," said N6 Tec Cofounder and CEO Pranav Patel. "Effectively, it's a real-time PCR thermal cycler where each individual well is completely independently controlled."
According to Patel, who cofounded N6 Tec in 2020, IconPCR was developed by the company "from the ground up" to help address the bottleneck of under- or over-amplifying libraries during PCR reactions while aiming to simplify the library prep workflow.
The platform, which is currently produced by N6 Tec’s contract manufacturing partner, will start shipping during the first quarter of this year and has a list price of $99,000.
Prior to N6 Tec, Patel was the founder and CEO of 2D Genomics, which was later acquired by Bio-Rad Laboratories, and has worked at genomics companies including Pacific Biosciences and 10x Genomics.
Roughly the size of a microwave oven, the IconPCR works in way almost identical to traditional PCR thermocyclers. Users can add their own PCR reaction mix to a 96-well plate or a strip tube, which is then loaded onto the platform for amplification.
However, instead of defining cycle numbers for the samples, as is the case for traditional thermocyclers, Icon PCR allows users to set the desired DNA output for each well while it automatically quantifies the amount of the starting material, Patel noted. Once the desired DNA concentration is reached in a particular well, the platform will automatically stop the PCR reaction and put the PCR products on hold.
N6 Tec did not publicly disclose the inner workings of the IconPCR in great detail. Patel noted that the platform, which is semiconductor-based, has an optical component and a fluorescent dye to help quantify the DNA in each well. Reaction volumes are between 10 microliters and 100 microliters per well.
Additionally, the drawer for loading the sample plate or strip is automation-compatible, enabling liquid handling robots to load samples onto the platform.
According to Patel, one advantage of IconPCR is that the platform allows users to "amplify all of the samples to the most optimum amount" while sparing them the quantification and normalization steps.
He considers the IconPCR technology beneficial for high-value or low-quality samples, such as cell-free DNA, ancient DNA, and formalin-fixed paraffin-embedded (FFPE) samples, which are often prone to overamplification because of low input or sample quality issues.
Additionally, Patel said IconPCR can be useful for PCR assay developers who desire to optimize their reaction conditions, given that the platform allows users to set up different reaction conditions in individual wells.
As for IconPCR’s data quality, Patel said there is "no drawback in terms of performance" compared to traditional thermocyclers.
During a company-sponsored workshop here at the Advances in Genome Biology and Technology annual meeting on Wednesday, N6 Tec showed some preliminary benchmarking data for IconPCR.
In one experiment, the company performed full-length 16S sequencing from 32 soil samples using libraries generated from both conventional PCR and IconPCR. Overall, IconPCR showed a "severalfold increase" in unique sequences and accurate representation of microbial population diversity.
In another experiment, the company analyzed 10x Genomics 3' single-cell RNA libraries prepared using traditional PCR versus IconPCR and showed that the single-cell analysis profiles were "virtually identical." Additionally, spatial profiles generated by 10x Visium using libraries prepared by conventional PCR versus IconPCR were "indistinguishable," according to the firm.
So far, N6 Tech has been working with a handful of early customers to help evaluate the IconPCR platform, Patel said.
"For me, the advantage of the IconPCR platform is the ability to amplify samples to sufficient levels without a priori knowledge about the sample input concentration or quality," Eric Chow, director of the University of California, San Francisco’s Center for Advanced Technology, said in an email.
"One example in the past is metagenomic sequencing of clinical samples like cerebrospinal fluid or ocular fluid where there is not enough material to even quantitate," he said. "We amplified samples with a set number of PCR cycles that worked for most samples but ended up overamplifying many. This can cause skews in the representation but also [in] overamplification bubble products that make library size selection more challenging."
Additionally, Chow said he sees benefits of IconPCR for damaged nucleic acids such as in FFPE samples as well as for spatial applications such as those using the 10x Visium platform, where it is recommended to take an aliquot of each sample for qPCR to determine the optimal PCR cycle number. "With IconPCR, this isn't required, and you don't need to waste precious samples in a separate qPCR step," he noted.
Chow said his lab first learned about the IconPCR technology last summer and has been talking to N6 Tec since then. So far, the team has brought a few dozen single-cell libraries and SARS-CoV-2 genomic samples to N6 Tec’s site and ran these samples on a "near-final production instrument" from the company.
Overall, the cycle times "seem no different" than those of other PCR machines, Chow said, adding that he does not "see any difference in the input or setup requirements" of IconPCR compared with traditional thermocyclers.
"We will definitely look into acquiring a system, depending on cost," Chow said. "I think there are more exciting applications we can develop by leveraging the real-time and independent thermocycling capabilities of the system."
Although more expensive than a conventional thermocycler, the platform had some conference attendees excited.
"If it works as described, I will buy one," said Bruce Kingham, a core lab director at the University of Delaware. “We process many highly multiplexed projects; it could pay for itself in two or three years."
It also remains to be seen how the IconPCR can be utilized for high-throughput applications, given it can process a maximum of 96 samples per run.
While the company currently focuses on NGS library preparation as the main application, the N6 Tec management team emphasized that the technology could be potentially useful for other applications such as protein analysis, qPCR, as well as other fluorescence-based analytical assays.
Based in Pleasanton, California, N6 Tec currently employs just under 20 people, Patel said. The company has raised an undisclosed amount of Series A funding, including investment from Telegraph Hill Partners.
With the money raised so far, Patel said the firm plans to "aggressively" grow its commercial team and to engineer future products, which he did not specify.