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Startup Samplix Developing Enrichment Technology to Amplify Target Sequence from Background DNA

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NEW YORK (GenomeWeb) – Samplix, a Denmark-based startup that formed in December 2013 as a spinout from renewable biofuels company Estibio, is developing an enrichment technique to amplify a target sequence from the background DNA.

The technique, probability directed isolation of nucleic acid sequences, or PINS, relies on serial dilutions and partitioning of a sample followed by multiple displacement amplification to isolate and amplify the target. In a proof of concept study published in PLoS One, the Samplix researchers demonstrated they could isolate a virus present in human DNA at a concentration of just one in 100,000 complete human genomes.

Samplix consists of just four employees, Marie Just Mikkelsen, chief technology officer, told In Sequence. The company was spun out of the renewable biogas company Estibio after realizing that some of the genetic engineering technology the researchers were developing could have broader applications, she said.

The firm's first product will be the enrichment technology, PINS. To demonstrate the technique, the researchers combined DNA that contained the human papilloma virus in a mixture of background human DNA without the virus at a ratio of 1 in 100,000. Then, they dilute and partition the entire sample into wells and amplify the contents in each well. The dilution/amplification is repeated until less than one out of five to 10 wells contain the target DNA molecule.

In order for the technique to work, the target must contain a known sequence that is detectable in the original sample and can be amplified with PCR. For instance, in the study, the researchers created PCR primers to correspond to a region of between 100 and 130 bp of human papilloma virus and were able to amplify and sequence the virus, which was in a sample at just one copy per 100,000 human genomes.

Mikkelsen said that the technology could be applied to really complex samples, such as identifying tumor DNA present at a low abundance in a sample of mostly normal DNA, or to identify a specific bacterium or virus in a large amount of background human DNA.

She said that the company is currently working with a number of early access users and plans to commercialize a first iteration of the enrichment technique by the beginning of 2015. In addition, she said the company is already working on a second version that will automate the process in order to decrease turnaround time and hands-on time.

Mikkelsen described PINS as a sieve. "We hold back the target and let the rest slip through," she said, so that "what is left [has] a higher concentration of the target. And then we amplify that and repeat the procedure until you have the enrichment that you need."

The main advantage of the technology is that "it can enrich for really low levels of target DNA compared to the total," she said. In addition, it is not necessary to know the entire sequence of the target, just about 40 bp, which allows for sequencing about 10 kb to 40 kb of flanking sequence — "an advantage when targeting a virus you don't know very well," she said.

In the study, the team demonstrated that they could enrich a sample containing HPV18 more than 275,000-fold through six rounds of enrichment. Sanger sequencing enabled the sequencing of a 3.2 kb fragment, however, the researchers reported that the enriched fragment is likely longer and could possibly be sequenced in its entirety with next-generation sequencing. In addition, data analysis enabled the researchers to detect the viral integration site.

Despite being subjected to so many rounds of enrichment, which can introduce errors, the sequences from the pure sample and the enriched sample were 100 percent identical, the authors reported. The authors attributed this to the use of the Phi29 enzyme for amplification, which has a low error rate.

Currently, Mikkelsen said the main limitation of the technique is the lack of automation in the protocol — something the company is working on improving. Right now, she said that users could enrich one sample between 5x and 10x in one day. But with automation, that will increase to 10,000-fold enrichment.

"At this point, the technique is really for people who need to do something that's currently not possible" with available technology, she said. With automation, the technique could have applications in infectious disease, oncology, and even prenatal testing, she said.

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