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Combining Nanopores and SBH, Brown U Spin-Off Aims for Long, Low-Cost Reads

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A new firm formed from the merger of two Brown University startups plans to develop a DNA sequencing technology that generates long reads at low cost, the company said last week.
 
Late last month one of the firms, NABsys, acquired the other, GeneSpectrum, in an all-stock transaction.
 
The combined company, which currently has three full-time employees, is developing hybridization-assisted nanopore sequencing, dubbed HANS, which combines sequencing-by-hybridization and nanopore sequencing. Both approaches were independently pursued by researchers at Brown, which spun off the two shops.
 
The merger “came out of the realization that the two approaches … were unlikely to finally win the day” on their own, but seem to have great promise as a pair, NABsys CEO Barrett Bready told In Sequence last week.
 
The goal is to be able to sequence a continuous 100,000-base stretch of DNA 18 months from now, and to develop an instrument within three to five years that can sequence a human genome “well below $1,000,” he said.
 
Eventually, Bready, who holds an undergraduate degree in physics and a medical degree from Brown, hopes the technology will enter personalized medicine by helping to analyze genetic predisposition to disease and cancer genomes.
 
NABsys’ other senior officer, John Oliver, vice president of research and development, headed GeneSpectrum as CEO and CSO. He founded GeneSpectrum in 2000 while he was an assistant professor in the chemistry department at Brown, together with two professors in the computer science department, Eli Upfal and Franco Preparata.
 
Armed with a grant of undisclosed size from the National Human Genome Research Institute and undisclosed state funding from the Slater Fund, GeneSpectrum set out to develop novel oligonucleotide probes for sequencing by hybridization, or SBH. These probes incorporate patterns of so-called universal bases, which can anneal to any of the four bases of DNA.
 
The patterned probes would allow researchers to use new types of algorithms for reconstructing the sequence of the target DNA based on the hybridization pattern of the probes, and “as a result, the amount of sequence information that you can get from one experiment is about two orders of magnitude higher than by the traditional sequencing-by-hybridization approach,” Oliver said last week.
 
Thus, scientists would be able to sequence 16,000 bases of DNA with a probe library of patterned 8-mers, instead of just 250 bases with a library of the same size made up of standard 8-mers.
 
However, “one of the big problems is, if a sequence appears twice in the piece [of DNA], if one of the oligonucleotides is complementary to two different places in that target sequence, then you lose track of where you were, and it makes it impossible to reconstruct the data,” Oliver said. “That is the primary reason that SBH just was not going to perform well.”
 
Enter nanopores. “The nanopore can in theory tell you the position that the probe is hybridizing to,” Oliver said. “So if you get two of them hybridizing on your piece of DNA, you know where they are.”
 
The idea is to thread a 100,000-base fragment of genomic target DNA that has multiple copies of a single probe attached to it through an electrically addressable nanopore and determine the position of the probes by the current blockade caused by the double-stranded DNA. Researchers can deduce the DNA sequence by repeating this process for every probe in the library and analyzing the resulting probe map.
 
This use of a nanopore circumvents the problem of resolving single bases, which may never become possible with nanopores, Bready said. “While it gets around the single-base resolution requirement for direct nanopore sequencing, it maintains intact the idea that nanopores inherently offer long read lengths,” he said.
 
The long reads will enable de novo sequencing, he said, and the method’s accuracy will be high because many probes interrogate each position on the unknown DNA.  
 
However, one nanopore is not sufficient for sequencing DNA in this way. “It was intuitively obvious that, because you are repeating this multiple times, it needs to be scaled,” Bready said. Therefore, NABsys’ first project, in collaboration with researchers at Brown, was to develop an addressable nanopore array.
 

“Making an addressable array is possible, the probe design and reconstruction algorithm is possible, so it’s really the implementation.”

NABsys was founded in 2004 by Xinsheng “Sean” Ling and Nobel laureate Leon Cooper, both professors of physics at Brown University. Ling has been developing nanopore technology for DNA sequencing and filed a patent application entitled “Hybridization-Assisted Nanopore Sequencing of Nucleic Acids” with the US Patent and Trademark Office in October 2006, according to his website. Another patent application, entitled “Adressable nanopores and micropores including methods for making and using same,” was published in 2005.
 
In 2004, Ling and three colleagues, one of them Amit Meller — who has been developing his own nanopore sequencing technology at Boston University — won a two-year, $1.5-million grant from the National Science Foundation for a project entitled “DNA Sequencing and Translocation Studies using Electrically-Addressable Nanopore Arrays.”
 
NABsys has exclusively licensed both the HANS technology and addressable nanopore arrays from Brown University.
 
The hybridization reaction products, Bready said, are each delivered to a different nanopore on the array using fluidics channels. The nanopore array is a silicon wafer, ultimately with thousands of pores, each of which is independently electronically addressable.
 
This year the company plans to figure out how precisely the nanopore detector can locate the position of the probes on the target DNA, and how that affects the performance characteristics of the technology. “Making an addressable array is possible, the probe design and reconstruction algorithm is possible, so it’s really the implementation” of the technology, Bready said.
 
“It often takes longer than you plan,” he said, “but we don’t see any fundamental obstacles that we worry we might not overcome.”
 
The company, which is supported by a state loan of undisclosed size from the Slater Technology Fund, also hopes to raise $1 million in a limited equity round this year.

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