SPECTRE-seq Single Primer Extension for Fragmented DNA Sequencing
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Solution Overview
Problem
Current methods for detecting and sequencing fragmented DNA, such as circulating tumor DNA (ctDNA) and HPV ctDNA, face limitations due to the requirement for opposing primer sequences, which are often not present in fragmented DNA, leading to incomplete capture and low sensitivity.
Innovation Solution
The SPECTRE-seq method employs a single primer extension technique with next-generation sequencing adapters and biotinylated nucleotides to generate libraries that can capture and sequence entire genomes, including HPV, by using a pool of target-specific forward primers that tile across the genome, enabling accurate detection and quantification of fragmented DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR-based approaches with two primers are used, then amplification of intact DNA is achieved, but detection of fragmented DNA is limited because primer binding sites may not both be present
Solution Approach 1:
The invention divides the traditional two-primer PCR approach into a single-primer extension method. Instead of requiring both forward and reverse primers to bind to intact DNA, the method uses one primer to extend across fragmented DNA, capturing sequences even when the DNA is broken into smaller pieces. This segmentation of the amplification process allows reliable detection of fragmented DNA while maintaining detection reliability.
Solution Approach 2:
The invention inverts the conventional PCR paradigm by using single-primer extension rather than two-primer amplification. This inversion allows the method to work with fragmented DNA where traditional PCR fails, as the single primer can bind to available sequences and extend across fragments, capturing viral DNA even when host DNA is fragmented and incomplete.
2Adaptability or versatility
If single primer extension techniques are used, then fragmented DNA detection is improved, but sensitivity and accuracy are reduced compared to conventional PCR
Solution Approach 1:
The invention introduces next-generation sequencing (NGS) adapters as intermediaries in the single-primer extension process. These adapters are incorporated during the extension reaction and enable subsequent NGS library preparation and sequencing. This intermediary step allows the method to maintain the adaptability of single-primer extension for fragmented DNA while achieving high sensitivity and accuracy through the power of NGS, resolving the trade-off between versatility and measurement precision.
3Manufacturing precision
If conventional library preparation methods are used, then sequencing of intact DNA is achieved, but capture of entire viral genomes from fragmented DNA is incomplete
Solution Approach 1:
The invention performs preliminary action by incorporating NGS adapters directly during the single-primer extension step, before library preparation and sequencing. This preliminary incorporation of adapters ensures that even fragmented viral DNA sequences are captured and prepared for sequencing, preventing information loss. The method proactively captures entire viral genomes by extending from multiple primer binding sites across the viral genome, even when DNA is fragmented, thereby maintaining manufacturing precision while minimizing information loss.
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
SPECTRE-seq significantly improves the sensitivity and accuracy of DNA detection and sequencing by allowing the capture of entire HPV genomes and other fragmented DNA, overcoming the limitations of conventional PCR methods and enabling more effective monitoring of HPV-associated cancers.
Implementation Method 1
performing a single primer extension reaction to generate first-generation copies of the target gene or genome of interest
Implementation Method 2
performing a nucleotide tailing reaction or adding a common sequence to the 3' end of the first-generation copies of the target gene or genome of interest
Implementation Method 3
performing a PCR reaction using: a forward primer complementary to a primer binding site within the target sequence, and a reverse primer complementary to the other primer binding site
Data Source
AI summary
The present invention provides modified single primer extension-based methods for generating an amplified library of fragments of a target gene or genome of interest from a sample of fragmented DNA, wherein the library is suitable for use in detecting, quantifying and/or sequencing the target gene or genome of interest. The present invention also provides compositions for use in such methods. In some embodiments the present invention provides methods and compositions specifically for detecting, quantifying and/or sequencing circulating tumor derived HPV DNA.