UMI-Tagged Genomic Copy Number Counting Without PCR Distortion
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Solution Overview
Problem
Current methods for obtaining genomic copy number information, such as whole genome amplification (WGA), suffer from over-sampling and non-uniform amplification, leading to distortions and limitations in single cell measurements, and are not suitable for single cell RNA profiling.
Innovation Solution
A method involving segmenting genomic material, tagging with unique tags, and performing PCR to generate tagged nucleic acid molecules, followed by sequencing and mapping to obtain copy number information without amplification distortion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If whole genome amplification (WGA) is used to obtain genomic copy number information, then the quantity of genomic material is increased, but amplification distortion and non-uniform sampling occur
Solution Approach 1:
The patent applies preliminary action by tagging each genomic DNA molecule with a unique molecular identifier (UMI) before any amplification occurs. This ensures that the tagging happens at the zero-th derivative stage, capturing the original molecular diversity before amplification distortion can occur. The UMI tags are incorporated into the DNA molecules during a preliminary tagging step, allowing subsequent amplification products to be traced back to their original parent molecules for accurate counting.
Solution Approach 2:
The patent uses unique molecular identifiers (UMIs) as intermediaries to bridge the gap between original genomic molecules and their amplification products. These UMI tags serve as mediators that carry information about the parent molecule through the amplification process, enabling accurate reconstruction of the original molecular population despite the distortion introduced by amplification. The UMI acts as a tracker that allows computational methods to correct for amplification bias.
2Quantity of substance
If PCR amplification is performed on tagged nucleic acid molecules, then the quantity of detectable molecules is increased, but amplification distortion may still occur
Solution Approach 1:
The patent implements feedback by using the UMI tags to monitor and correct for amplification distortion computationally. After amplification, the sequencing data is processed to count only unique UMIs at each genomic location, and the frequency distribution of amplification products is analyzed to detect and correct for bias. This feedback loop allows the system to identify and compensate for regions that were over- or under-amplified, restoring reliability to the copy number measurements.
3Measurement precision
If ligation-mediated PCR is used to avoid WGA distortion, then amplification distortion is reduced, but the method still requires an amplification step and has limited applicability
Solution Approach 1:
The patent achieves universality by developing a tagging and counting methodology that can be applied to multiple nucleic acid types and experimental designs. The UMI tagging approach works for genomic DNA, mRNA, and other nucleic acid targets. The method is compatible with various amplification strategies including PCR, WGA, and ligation-mediated amplification, making it universally applicable across different sample types and experimental conditions while maintaining accuracy.
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 accurate determination of genomic copy numbers without amplification distortions, suitable for complex samples and high-throughput sequencing.
Implementation Method 1
subjecting the zero-th order derivative strands to a polymerase reaction in the presence of essentially unique primers... thereby generating first order derivative strands; adding a polynucleotide tail to the first order derivative strands; subjecting the first order derivative strands to a polymerase reaction... thereby generating second order derivative strands
Data Source
AI summary
A method for obtaining from genomic material genomic copy number information unaffected by amplification distortion, comprising obtaining segments of the genomic material, tagging the segments with substantially unique tags to generate tagged nucleic acid molecules, such that each tagged nucleic acid molecule comprises one segment of the genomic material and a tag, subjecting the tagged nucleic acid molecules to amplification by polymerase chain reaction (PCR), generating tag associated sequence reads by sequencing the product of the PCR reaction, assigning each tagged nucleic acid molecule to a location on a genome associated with the genomic material by mapping the subsequence of each tag associated sequence read corresponding to a segment of the genomic material to a location on the genome, and counting the number of tagged nucleic acid molecules having a different tag that have been assigned to the same location on the genome, thereby obtaining genomic copy number information unaffected by amplification distortion.