Finally a nanopore sequencer that works

Today I was given an exclusive preview from the newest nanopore sequencing company on the planet, Norfolk Nanopore Technology. The new "Polonopore"technology has been incubating in the Norwich Research Park BioIncubator under the same roof as TGAC.

NNT's brand spanking new nanopore sequencer could mean the end for the HiSeq, MiSeq, Proton and PGM platforms and paves the way for $100 personal genomes. NNT are suggesting very high yields from long-reads using Polonopore technology. $100 for 100x coverage of 100 genomes in 100 hours.



NNT's nanopore seqeuncer: There has been rampant speculation that other groups are looking into nanopores of their own after ONT's strand-sequencing announcements at AGBT in February 2012. Jay Flatley (Illumina's CEO) also made some mention of Illumina's own nanopore progress at this years JP Morgan conference.

The NNT Polonopore is a hybrid system, melding the very best of current NGS technology with the sensitivity, read-length and throughput of a nanoscale sequencer. They have worked with multiple players to bring the Polonopore instrument to market, collaborating on different aspects of the technology.

How does it work or why bother with single-molecules: NNT has been thinking out of the box and their hybrid technology does not throw out the past five years of work in improving NGS library preparation, cluster/bead/polony amplification of signal or cycle-sequencing. Their box quite clearly has a Solexa sequencer pedigree, although the optics are gone and fluidics for sequencing vastly simplified. The new system is similar in concept to Illumina's a cBot, but also conceptually similar to bits of ONT, Ion Torrent or DNA electronics. It can take flowcells of varying size making the price of sequencing dependant on run length and the number of reads generated.

NNT build polonies directly over nanopores; cluster growth is defined by photolithograhic placement of the "seq-cell" oligos (Fig1:1). Polonies are built very similarly to the original Manteia "DNA colony sequencing" technology then linearised (Fig1:2-4). Now comes the clever bit; a reversibly-lockable exonuclease is added which binds to the 3' end of the DNA and removes the first base of the adaptor. A laser "unlocks" the exonuclease allowing a single base to be cleaved and to translocate through the pore (Fig1:5-6a/b). Immediately upon cleaving the DNA the exonuclease is locked preventing more than a single base excision. Repeated pulses of laser-activation allow each base to be read off with no further fluidics, no chemistry and very quickly. Each base gives a characteristic signature on the electrical readout from each pore (Fig1:7) and the unlock-cleavage-lock cycle is performed at a conservative 1 base per second generating 15kb in a little over four hours.

Figure 1 from the NNT "Polonopore" patent

Around 1000 nulceotides are released in each cycle, one from each molecule in the polony, this amplifies signal making detection more robust. And because this is an enzymatic reaction with no chemistry only one base can ever be cleaved making phasing and prephasing almost zero, allowing longer reads. Long-range PCR polonies are allowing read lengths of up to 15kb, using a universal long-range PCR on adaptor modified large fragments of DNA.

Can NNT crack the $100 genome: With between 100,000 and 4M nanopores per chip 1.5Gb-60Gb are available today, although no pricing was announced for the instrument. Increasing the cycling to 10bp/sec and pores to 10M allows 100x 100-fold coverage genomes in 100 hours. as long as the chips don't cost too much then the $100 genome seems to be pretty close.

Other nanopore developers have made much of library-free, single-molecule approaches and these may well be the systems we are using in 5, 10 or 15 years time. However no-one has yet developed a robust system capable of generating large amounts of data from complex genomes.

NNT's strategy of bringing the best of today and what is possible from tomorrow's technology may be seen as flawed by some. The results will speak for themselves though as early access users start to present data. Very few users have a need for true single molecule sequencing and most are already comfortable with current library prep and PCR-amplification methods.

Roll on NNT. I can't wait to get mine on April 1st 2014.