Nanopore Sequencing via Raman Spectra for Long DNA Assembly
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in accurately assembling repeated regions, correctly placing segments within haplotypes, and performing de novo assembly without a reference genome, particularly due to limitations in read length and phase sensitivity.
Innovation Solution
The development of a nanopore-based long-read sequencing method using nanochannel chips with tortuous nanopores and electromagnetic-field enhancement structures for single-base resolution, enabling the direct detection of nucleic acid bases through optical techniques like SECARS without labeling, and allowing for the sequencing of long-chain molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If short-read shotgun sequencing is used, then high throughput and parallel processing are achieved, but inability to correctly piece together repeated regions and associate segments with specific chromosomes occurs
Solution Approach 1:
The invention divides the genome into long continuous reads rather than short fragments. By sequencing entire long-chain DNA molecules (kilobase to megabase scale) as single continuous sequences, the method segments the assembly problem into manageable long reads that can be uniquely placed in the genome, resolving repeated regions through their unique flanking sequences.
Solution Approach 2:
The invention transitions from one-dimensional short reads to multi-dimensional long reads by incorporating spatial information through nanochannel translocation. The physical position and translocation kinetics of DNA molecules through nanochannels provide additional dimensional data that enables accurate assembly and phasing of genetic variants across chromosomes.
2Reliability
If reference genome-based assembly is used, then sequencing coverage is improved, but de novo assembly without reference genome becomes difficult
Solution Approach 1:
The invention enables self-service assembly by generating sufficiently long reads that contain unique sequence information for self-alignment and de novo assembly. The long reads inherently provide enough contextual information to assemble genomes without reference, while still allowing reference-based approaches when desired, making the system adaptable to both modes.
3Measurement precision
If optical detection techniques are used for single-base resolution, then labeling requirements increase complexity, but label-free detection is challenging
Solution Approach 1:
The invention extracts and removes the labeling step from the sequencing process by using label-free optical detection. By employing techniques such as surface-enhanced Raman scattering (SERS) or direct optical absorption/dichroism of the DNA bases themselves, the method achieves single-base resolution without requiring fluorescent or other labels, thereby reducing chemical complexity and potential artifacts.
4Measurement precision
If long-chain molecules are sequenced, then repeated regions and haplotype phasing are resolved, but translocation speed control becomes critical
Solution Approach 1:
The invention applies dynamic control of translocation speed through adjustable electric fields across the nanochannels. By modulating the voltage and using nanochannel geometry (length, width, surface charge), the system optimizes translocation velocity to ensure complete bases pass through the detection zone at resolvable speeds while maintaining high throughput, adapting to different DNA lengths and sequences.
Data Source
AI summary
Nucleic acid sequencing methods and systems, the systems including nanochannel chip including: a nanochannel formed in an upper surface of the nanochannel chip and; a roof covering the nanochannel and comprising nanopores and a field enhancement structure; and a barrier disposed in the nanochannel. The method including: introducing a buffer solution including long-chain nucleic acids to the nanochannel chip; applying a voltage potential across the nanochannel chip to drive the nucleic acids through the nanochannel, towards the barrier, and to translocate the nucleic acids through nanopores adjacent to the barrier, such that bases of each of the nucleic acids pass through the field enhancement structure one base at a time and emerge onto an upper surface of the roof; detecting the Raman spectra of the bases of the nucleic acids as each base passes through the electromagnetic-field enhancement structure; and sequencing the nucleic acids based on the detected Raman spectra.