Digitized Transposons for Haploidome Determination
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Solution Overview
Problem
Current methods for whole genome amplification, such as PCR-based or isothermal amplification, lead to biased results, making it difficult to determine copy number variation (CNV) and single nucleotide polymorphisms (SNPs) accurately, especially in complex cells like cancer cells where chromosome numbers vary significantly and ultra-rare mutations are challenging to identify due to high error rates in sequencing.
Innovation Solution
The use of digitized transposons to introduce uniquely barcoded transposons into genomic DNA, allowing for the unambiguous identification of alleles and copy numbers by generating distinct patterns that distinguish between genuine variations and sequencing errors, thereby facilitating accurate haploidome determination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PCR-based or isothermal amplification methods are used for whole genome amplification, then amplification of genomic DNA is achieved, but biased amplification occurs resulting in over-amplification of some areas and under-amplification of others, making copy number variation determination and SNP identification difficult
Solution Approach 1:
The patent introduces transposons as intermediary elements that randomly integrate into the genome before amplification. These transposons serve as molecular beacons that mark specific genomic locations independently of amplification bias. By counting transposon integrations rather than relying on amplification signal intensity, the method accurately determines copy number variations even when conventional amplification methods produce biased results
Solution Approach 2:
The patent creates multiple copies of transposon-marked genomic regions through controlled transposition events. Each transposon integration event creates a permanent molecular record of the original genomic copy number. This copying mechanism bypasses amplification bias because the transposon integrations occur before and independently of the amplification process, preserving accurate copy number information
2Productivity
If conventional sequencing methods are used to identify SNPs and mutations, then sequencing data is obtained, but the typical error rate of ∼1% results in hundreds of millions of sequencing mistakes that become problematic in identifying ultra-rare mutations
Solution Approach 1:
The patent uses transposons as intermediary markers that physically link to specific genomic loci. Each transposon carries a unique molecular identifier that serves as a reliable reference point. By associating sequencing reads with their corresponding transposon markers, the method can distinguish true rare mutations from sequencing errors, as the transposon marker provides an independent verification system that is not subject to the same error rates as conventional sequencing
3Ease of manufacture
If computer programs operate on the assumption that each genome consists of 44 euchromosomes and two sex chromosomes, then standard analysis can be performed, but this assumption does not hold for all cells and certainly not for cancer cells which exhibit vast differences in copy number
Solution Approach 1:
The patent fundamentally changes the analytical parameter from assuming a fixed diploid chromosome structure to measuring actual transposon integration counts at each genomic locus. This parameter change allows the method to adapt to any ploidy level or chromosomal abnormality, including cancer cells with complex karyotypes. The transposon count directly reflects the true copy number regardless of whether the cell is diploid, triploid, tetraploid, or has chromosomal rearrangements
Solution Approach 2:
Instead of assuming a standard genome structure and trying to fit data to that model, the patent inverts the approach by using transposon integration counts as the primary measurement and deriving genome structure information from those counts. This inversion allows the method to accurately analyze any genome configuration without requiring prior assumptions about chromosome number or structure
4Measurement precision
If deep sequencing is used to detect rare mutations in cancer studies, then increased sensitivity is achieved, but the typical error rate still results in numerous sequencing mistakes that remain problematic
Solution Approach 1:
The patent introduces transposons as intermediary molecular markers that provide an independent verification system. Each transposon integration event creates a unique molecular barcode that can be used to track and verify sequencing reads. This intermediary system allows the method to distinguish true rare mutations from sequencing errors even at low frequencies, because the transposon marker provides an additional layer of information that is not subject to sequencing errors
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
This approach enables clear identification of copy number variations and SNPs, differentiates between homozygotes and heterozygotes, and determines linked loci, improving the accuracy of haploidome analysis in complex cellular environments by overcoming amplification biases and sequencing errors.
Implementation Method 1
contacting the sample nucleic acids with a loaded transposase capable of incorporating transposons into the sample nucleic acids
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3B
AI summary
In certain embodiments, the present invention provides a way of "digitally" marking different the alleles of different chromosomes by using a transposase to insert differently barcoded transposons into genomic DNA before further analysis. According to this method, each allele becomes marked with a unique pattern of transposon barcodes. Because each unique pattern of transposon barcodes identifies a particular allele, the method facilitates determinations of ploidy and copy number variation, improves the ability to discriminate among homozygotes, heterozygotes, and patterns arising from sequencing errors, and allows loci separated by uninformative stretches of DNA to be identified as linked loci, thereby facilitating haplotype determinations. Also provided is a novel artificial transposon end that includes a barcode sequence in two or more positions that are not essential for transposition.