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.
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.
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.
For more information, please visit www.nanostring.com.
news release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934 and the Private Securities Litigation Reform Act of 1995. These forward-looking statements include statements regarding the expected performance of the company's Hyb & Seq chemistry and its potential superiority to existing sequencing methods, the company's Hyb & Seq chemistry's potential role as the basis for development of a DNA-sequencing instrument, the commercial potential of the company's Hyb & Seq chemistry and the anticipated timing for release of additional data. Such statements are based on current assumptions that involve risks and uncertainties that could cause actual outcomes and results to differ materially. These risks and uncertainties, many of which are beyond our control, include market acceptance of our
products; delays or denials of regulatory approvals or clearances for products or applications; delays or denials of reimbursement for diagnostic products; the impact of competition; the impact of expanded sales, marketing, product development and clinical activities on operating expenses; delays or other unforeseen problems with respect to manufacturing, product development or clinical studies; adverse conditions in the general domestic and global economic markets; as well as the other risks set forth in the company's filings with the
Leigh Salvoof Westwicke Partnersleigh.firstname.lastname@example.org 415-513-1281
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