Sanger sequencing trace trimming for indel detection
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Solution Overview
Problem
Current methods for assessing nucleic acid editing outcomes, such as those using CRISPR/Cas systems, are often costly and time-consuming, and may not be readily available, particularly for detecting insertions and deletions in nucleic acid sequences.
Innovation Solution
A method involving a computer system that determines predicted mutated sequencing traces from Sanger sequencing data to deduce nucleic acid editing outcomes, using regression analysis and trimming techniques based on guide sequences to identify and report predicted sequences and their frequencies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If massive parallel sequencing technique is used to detect nucleic acid editing outcomes, then measurement precision is improved, but cost and time consumption increase
Solution Approach 1:
The patent extracts and analyzes only the specific regions of interest (putative indel regions) from the full sequencing data. By focusing computational analysis on trimmed sequences around predicted cut sites rather than processing entire genomes, the method achieves accurate indel detection while significantly reducing computational time and resource requirements compared to comprehensive massive parallel sequencing analysis.
Solution Approach 2:
The patent segments the sequencing analysis into distinct steps: (1) identifying putative cut sites based on guide sequences, (2) trimming sequences to focus on regions around cut sites, (3) generating predicted mutated sequences, and (4) comparing to actual sequencing data. This segmentation allows the method to achieve high measurement precision for indel detection while reducing overall analysis time through focused processing of critical regions only.
2Measurement precision
If massive parallel sequencing technique is used to assess nucleic acid editing outcomes, then measurement precision is improved, but availability decreases
Solution Approach 1:
The patent creates a computational model that replicates the essential function of massive parallel sequencing by using Sanger sequencing data combined with in silico analysis. Instead of requiring actual NGS infrastructure, the method copies the analytical capability through bioinformatics processing of simpler sequencing data, making the technology widely available without specialized equipment while maintaining detection precision through sophisticated algorithms.
Solution Approach 2:
The patent introduces computational analysis as an intermediary between Sanger sequencing and the detection of indels. Rather than directly using NGS technology, the method employs bioinformatics processing (sequence trimming, predicted mutation generation, regression analysis) as a mediator to extract precise indel information from simpler sequencing data, thereby achieving NGS-level precision with more accessible technology.
3Quantity of substance
If Sanger sequencing with computational analysis is used instead of massive parallel sequencing, then cost is reduced, but measurement precision may worsen
Solution Approach 1:
The patent changes the analytical parameters by applying regression analysis with quality thresholds (R-squared values, p-values) to the Sanger sequencing data. This statistical parameter transformation allows the method to extract precise indel detection capability from lower-resolution Sanger data, maintaining measurement precision comparable to NGS while reducing costs through the use of cheaper sequencing technology combined with sophisticated computational analysis.
Data Source
AI summary
The present disclosure provides a method for analyzing nucleic acid sequences. The method can comprise determining, by a computer system, a base trace by trimming a Sanger sequencing trace of a plurality of nucleic acid molecules from a sample based on a first target sequence and a second target sequence. Each of the first and second target sequences can be in the plurality of nucleic acid molecules or can be in the complement of sequence of the plurality of nucleic acid molecules.