Nucleic Acid Sequencing Preparation with Target-Specific Priming
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid enrichment methods for next-generation sequencing are costly, time-consuming, labor-intensive, and suffer from low specificity and off-target sequencing, particularly in hybridization-based capture assays.
Innovation Solution
A method involving target-specific priming, template-dependent extension reactions, and amplification using tailed random primers with nested sequencing primers to enrich and sequence nucleic acids, allowing for high specificity and sensitivity in determining contiguous nucleotide sequences, especially for gene rearrangements like fusion oncogenes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization-based capture assays are used for target enrichment, then sequencing coverage is achieved, but off-target sequencing occurs and specificity is reduced
Solution Approach 1:
The method segments the enrichment process into two distinct stages: (1) target-specific primer extension that precisely defines the enrichment boundary, and (2) tailed random primer extension that sequences only the unknown region adjacent to the target. This segmentation prevents off-target sequencing by eliminating the need for broad hybridization capture.
Solution Approach 2:
The target-specific primer extension is performed as a preliminary action before random sequencing. This preliminary step establishes precise boundaries by extending from the known target sequence into the unknown region, thereby defining exactly which sequences will be subjected to random priming and sequencing, thus preventing off-target enrichment.
2Productivity
If hybridization-based capture assays are used, then target enrichment is achieved, but the process becomes time-consuming and labor-intensive
Solution Approach 1:
The method merges the enrichment and sequencing preparation steps into a single integrated PCR reaction. The target-specific primer and tailed random primer work together in one amplification reaction to both enrich the target and prepare it for sequencing, eliminating the need for separate hybridization capture and washing steps required by conventional methods.
Solution Approach 2:
The tailed random primers self-selectively bind to the extended target sequences generated in the first extension step. The random portion of these primers anneals to the unknown region while the tail portion provides the sequencing adapter sequence, allowing the system to automatically prepare sequencing-ready products without additional manual intervention or complex instrumentation.
3Productivity
If conventional enrichment methods are used, then sequencing is performed, but cost-effectiveness is reduced
Solution Approach 1:
The method uses simple, inexpensive PCR reagents and primers instead of costly hybridization capture probes and specialized equipment. The tailed random primers are inexpensive synthetic oligonucleotides that can be easily synthesized, and the entire process uses standard PCR thermocyclers already present in most laboratories, eliminating the need for expensive specialized enrichment instruments.
4Measurement precision
If hybridization-based capture is used, then target sequences are enriched, but sensitivity for low-frequency mutations is reduced
Solution Approach 1:
By segmenting the enrichment to focus only on the specific unknown region adjacent to the target sequence rather than broad hybridization capture, the method concentrates sequencing depth on the region of interest. This increased local coverage improves the ability to detect low-frequency mutations through higher allele counting statistics.
Solution Approach 2:
The target-specific primer extension serves as a preliminary enrichment step that concentrates all target molecules at the specific locus before random sequencing begins. This preliminary concentration ensures that even low-abundance target sequences are adequately represented in the final library, improving detection sensitivity for rare mutations.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient and specific sequencing of contiguous nucleotide sequences, particularly for gene rearrangements, with improved sensitivity and coverage, facilitating detection of low-frequency mutations and gene fusions.
Implementation Method 1
contacting a target nucleic acid molecule comprising the known target nucleotide sequence with an initial target-specific primer under hybridization conditions
Implementation Method 2
performing a template-dependent extension reaction that is primed by a hybridized initial target-specific primer and that uses the target nucleic acid molecule as a template
Implementation Method 3
contacting the product of step (b) with a population of tailed random primers under hybridization conditions
Data Source
AI summary
Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.