Feb 15 2016
NanoString Technologies, Inc., a provider of life science tools for translational research and molecular diagnostic products, today presented the first proof-of-concept data for its novel massively parallel single molecule sequencing chemistry, Hyb & SeqTM, at the Advances in Genome Biology and Technology (AGBT) Meeting being held in Orlando, Florida. Hyb & Seq chemistry is based on NanoString's proprietary optical barcoding technology with several unique features giving it the potential to provide highly accurate data with a simple, efficient workflow.
The new chemistry was presented in a poster titled, "Enzyme-free, Amplification-free, Hybridization-based Single Molecule Sequencing (Hyb & SeqTM)," the abstract for which is provided below.
The Hyb & Seq chemistry is designed to enable a workflow that is simpler and faster than current sequencing methods due to the absence of library preparation, enzymes and amplification, while providing both short and long read capability. In proof-of-concept experiments, Hyb & Seq chemistry demonstrated a low intrinsic error rate, and the ability to provide high consensus accuracy at low coverage by non-destructively sequencing the same native DNA multiple times. Chad Nusbaum, Ph.D., a member of NanoString's Sequencing Advisory Board, commented, "I'm very excited about the potential of the Hyb & Seq chemistry. The combination of simple workflow and low error rate make it a promising basis for the development of a targeted sequencing system that would be well positioned for clinical applications."
As described in the poster, Hyb & Seq works through four basic steps. First, native DNA molecules from regions of interest are captured on a flow cell. Second, the captured DNA is washed with a mixture of probes containing fluorescent optical barcodes that hybridize to matched regions along the target DNA molecule. Third, the optical barcodes of the hybridized probes are read to identify both the base and position identities. Finally, the probes are washed away and the cycle is then repeated until the targeted regions have been read sufficiently to assemble the DNA sequence.
The proof-of-concept data was achieved using a prototype sequencer based on a modified nCounter SPRINTTM Profiler instrument and mixtures of synthetic DNA targets, including the BRAF V600E model system. The raw single pass error rate was measured at approximately 2% for a single target and between 2% and 4% for mixtures of ten targets. Joe Beechem, Ph.D., Senior Vice President of R&D for NanoString, commented, "Hyb & Seq is a natural extension of the company's optical barcoding technology, potentially providing another important dimension to our 3D BiologyTM applications. While potential commercialization of the chemistry is still several years away, we will continue to focus on development of the chemistry, and expect to present further data later this year."
Presentation Abstract
Hyb & SeqTM is an enzyme-free, amplification-free, single molecule sequencing chemistry using cyclic nucleic acid hybridization of fluorescent molecular barcoded n-mers. Targets are sequenced using barcodes with the structure:(nnb1b2b3b4b5b6nn—linker—r1r2r3r4r5r6), where: n = mixture of all 4 bases, b1 through b6 are the 6 contiguous bases that form the complement to the target DNA/RNA to be sequenced, and r1 through r6 is a sub-diffraction-limited cyclically-read optical barcode that encodes the identity of the six-bases hybridized to that target via 4-color fluorescence (~25 fluorophores per "r").
Genomic DNA or RNA is first "gapped" to generate a single-stranded region and captured onto a flow-cell. Optical barcodes are hybridized to these single-molecule targets, and bases at each hybridized target are read through a series of 6 "fast-exchange" hybridizations to the reporter (r)-regions. Each cycle yields a 6-base read. The hybridized probes are eluted, and the cycle is repeated until all regions have sufficient coverage.
Proof-of-concept was achieved using a prototype sequencer based on a modified nCounter Sprint Profiler instrument and BRAF V600E model system. Fast reporter readout-cycling was confirmed ( < 10 seconds). The raw single-pass error rate averaged 2.1% (min = 0.66%, max = 3.72%) and did not reveal any systematic bias. Based on this data, less than 5x coverage from a single molecule would be required to reach a consensus sequence accuracy of 99.99% (Q40).
Hyb & Seq has many potential advantages over other sequencing chemistries. Read lengths can be as long as the gapped single-stranded regions. Sequencing error is independent of read length. No covalent bonds are formed, making each cycle non-destructive and allowing multiple reads on the same native single molecule, drastically reducing error rates. The workflow does not require enzymes, amplification, or libraries, opening the possibility of a sample-to-answer sequencing instrument appropriate for clinical use.
Source: http://www.nanostring.com/