Multiplex SNP Sequencing for Low-Level Semen Sample Contamination
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Solution Overview
Problem
Existing methods for evaluating genetic purity in samples, such as animal semen straws, are insufficient for detecting low levels of contamination and mislabeling, particularly when samples are non-uniform, as they often rely on uniform samples and cannot confirm a 100% genetic match.
Innovation Solution
A multiplex sequencing method using SNP primer pairs to amplify and sequence genetic samples, followed by analysis to determine allele frequencies and compare them to reference sequences, allowing for the detection of contamination and confirmation of genetic identity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing microarray or chip assays are used to evaluate genetic purity, then the evaluation process is simple and fast, but the detection precision is insufficient for low levels of contamination
Solution Approach 1:
The patent segments the genetic sample analysis into multiple discrete SNP loci evaluations. Each SNP locus is independently analyzed for allele frequencies, allowing precise detection of contamination at specific genetic locations. This segmentation enables the system to identify even low-level contamination (1% or less) by examining individual SNP positions rather than treating the entire genome as a single unit.
Solution Approach 2:
The patent changes the measurement parameter from simple presence/absence detection to quantitative allele frequency analysis. By measuring the frequency of alternative alleles at SNP loci and comparing against expected frequencies, the system can detect contamination at very low levels. This parameter change transforms the detection capability from qualitative to quantitative, enabling precise measurement of contamination levels.
2Measurement precision
If multiplex sequencing methods are used on non-uniform samples, then contamination detection sensitivity increases, but the difficulty of detecting and measuring increases due to sample heterogeneity
Solution Approach 1:
The patent extracts and isolates specific SNP loci from the complex non-uniform sample matrix. By using targeted PCR amplification of selected SNP regions, the method separates the signal of interest (SNP alleles) from the background complexity of dead sperm, UV-irradiated sperm, and other sample heterogeneities. This extraction approach allows multiplex sequencing to achieve high sensitivity despite sample non-uniformity.
Solution Approach 2:
The patent introduces SNP-specific primers and adapters as intermediary molecules that facilitate the sequencing process. These intermediaries bind specifically to target SNP regions and enable universal amplification and sequencing protocols to work effectively on non-uniform samples. The intermediaries act as bridges between the heterogeneous sample components and the sequencing machinery, resolving the detection difficulty.
3Measurement precision
If existing methods functionally measure one allele per line, then the method complexity is low, but the measurement precision for determining genetic composition is insufficient
Solution Approach 1:
The patent adds a quantitative dimension to genetic analysis by measuring allele frequencies rather than simply detecting presence or absence. This dimensional change transforms the data from binary (one allele per line) to continuous (frequency values), enabling precise determination of genetic composition and detection of mixed samples. The frequency measurement adds information depth that reveals contamination and mixture proportions.
Solution Approach 2:
The patent creates a universal SNP panel that can evaluate multiple genetic loci simultaneously using the same amplification and sequencing protocol. This multi-functional approach allows a single assay to measure many SNPs across the genome, providing comprehensive genetic composition analysis. The universal primer set and sequencing workflow handle diverse SNP locations with equal effectiveness, increasing precision without proportionally increasing complexity.
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
The method enables accurate detection of contamination as low as 1% and confirms genetic purity, ensuring samples match the intended genetic material and preventing costly errors, meeting high purity standards.
Implementation Method 1
subjecting the extracted DNA from the test sample to nucleotide amplification using a pool of SNP primer pairs, each SNP primer pair flanking a unique locus that contains a single target SNP defining a first allele and a second allele, the nucleotide amplification produces amplicons for each SNP primer pair generating a pool of SNP amplicons
Implementation Method 2
subjecting the pool of SNP amplicons to next-generation sequencing (NGS) to generate a nucleotide sequence for each amplicon in the pool
Data Source
AI summary
Methods and systems for processing semen samples from straws for determining genetic identity, testing the purity of the sample, detecting errors or contamination, calculating an amount of contamination, and determining the identity of the contaminant. The methods herein can detect low levels of contamination, such as contamination of about 0.5%, 1%, 2%, etc.