DNA Quantification via Ligation Oligonucleotides and UMIs
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Solution Overview
Problem
Existing methods for quantifying DNA sequences face challenges such as unbalanced amplification leading to biased detection and lower sequencing coverage due to the transfer of only an aliquot of ligated products to the amplification step, resulting in allelic drop-out and PCR-induced bias.
Innovation Solution
A method involving contacting target DNA sequences with left and right ligation oligonucleotides, followed by ligation, immobilization, and amplification using unique molecular identifiers (UMIs) and barcodes to enable absolute molecule counting and maximize sequencing coverage, while minimizing PCR bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If an aliquot of ligated products is transferred to the amplification step, then the process is simplified, but sequencing coverage is reduced and allelic drop-out occurs
Solution Approach 1:
The patent applies preliminary action by performing ligation of oligonucleotides to target DNA sequences before the amplification step. This ensures that all target sequences are pre-assembled with their corresponding oligonucleotides, preventing allelic drop-out during subsequent amplification and maximizing sequencing coverage while maintaining process simplicity
2Measurement precision
If PCR amplification is used to multiply original nucleic acid molecules, then detection sensitivity is improved, but PCR-induced bias and unspecific amplification occur
Solution Approach 1:
The patent uses ligated oligonucleotide products as intermediaries between the target DNA sequences and the PCR amplification process. These pre-ligated products serve as specific templates that reduce unspecific amplification and PCR-induced bias while maintaining detection sensitivity, as they contain unique molecular identifiers that enable accurate quantification
3Measurement precision
If unique molecular identifiers are incorporated into ligation oligonucleotides, then absolute molecule counting is enabled, but the complexity of the assay increases
Solution Approach 1:
The patent uses unique molecular identifiers (UMIs) as digital copies or tags associated with each original DNA molecule. During amplification, these UMIs are replicated along with the target sequence, allowing bioinformatic tools to group and count reads by their UMI identifiers, thereby enabling absolute molecule counting without significantly increasing wet-lab procedural 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
This approach allows for accurate and absolute quantification of target DNA sequences with high specificity and sensitivity, ensuring maximum coverage of unique DNA molecules and eliminating unspecific amplification, thereby overcoming the limitations of prior art techniques.
Implementation Method 1
contacting, in each container of N separate containers, target DNA sequences, under hybridization conditions, with M L left ligation oligonucleotides and M R right ligation oligonucleotides
Implementation Method 2
adding, in each container of the N separate containers, a ligating agent capable of ligating together the 3' end of a left ligation oligonucleotide and the 5' end of a right ligation oligonucleotide while hybridized to a target DNA sequence to form a ligated product
Implementation Method 3
immobilizing, in each container of the N separate containers, the ligated product in complex with the target DNA sequence onto a solid phase having preference for DNA sequences
Implementation Method 4
amplifying, in each container of the N separate containers, the ligated product in presence of a left amplification primer and a right amplification primer to form an amplified product comprising two UMIs and one barcode sequence
Data Source
Figure 1A~1C
Figure 1D~1E
Figure 2~3
AI summary
A target DNA sequence (1 ) is contacted with ML+MR ligation oligonucleotides (10, 20) under hybridization conditions. The ligation oligonucleotides (10, 20) comprises a respective UMI (14, 15). A ligating agent ligates together the ligation oligonucleotides (10, 20) while hybridized to the target DNA sequence (1) to form a ligated product (30). The ligated product (30) is amplified by means to amplification primers (40, 50), of which one comprises a sample-specific barcode sequence (55), to form an amplified product (60) comprising two UMIs (65), a sequence of interest (66) and one barcode sequence (68). Amplified products (60) from multiple samples are pooled together, sequenced, demultiplexed and mapped to enable quantification of unique target DNA sequences (1) in the different samples.