Transposase Random Priming for Short Fragment DNA Sequencing
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Solution Overview
Problem
Existing transposase-based tagging methods struggle with sequencing small, fragmented, or chemically modified DNA samples, such as FFPE solid tumor samples and cell-free DNA, due to the requirement of two adjacent transposase insertions, which are often not feasible for short DNA fragments.
Innovation Solution
A method involving a single transposase adapter per DNA fragment, combined with adapter-tagging and primer extension reactions, followed by PCR amplification, to generate adapter-tagged and amplified DNA fragments suitable for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hybridization and PCR are used for DNA sample preparation, then amplification of target sequences is achieved, but the process is time-consuming and labor-intensive
Solution Approach 1:
The invention extracts and eliminates the time-consuming hybridization and PCR steps from the DNA sample preparation process by using transposase-random priming, which directly amplifies target sequences without requiring these intermediate steps
Solution Approach 2:
The transposase-random priming method performs preliminary fragmentation and tagging of DNA sequences during the initial setup, eliminating the need for subsequent hybridization and PCR amplification steps
2Productivity
If transposase-random priming is used, then DNA sample preparation time is reduced, but uniform coverage of the genome may be compromised
Solution Approach 1:
The invention changes the parameters of transposase activity and priming conditions to optimize both speed and coverage uniformity, achieving rapid DNA sample preparation while maintaining acceptable genome coverage
Solution Approach 2:
The method uses multiple transposase-random priming reactions with different conditions to ensure comprehensive genome coverage, compensating for any individual reaction's non-uniformity
3Measurement precision
If extensive DNA fragmentation and enrichment are performed, then target sequences are isolated, but DNA loss increases
Solution Approach 1:
The invention uses transposase-random priming as an intermediary method that directly tags and amplifies target sequences without requiring extensive fragmentation and enrichment steps, thereby reducing DNA loss while maintaining isolation 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
Enhances the capture and sequencing efficiency of short DNA fragments by allowing recovery of genomic DNA sequences on either side of the tagmentation site, overcoming limitations of conventional methods that require two transposase adapters.
Implementation Method 1
Transposase-random priming DNA sample preparation methods and compositions are disclosed herein.
Implementation Method 2
amplification by PCR, RPA, or other methods known in the art
Implementation Method 3
hybridization and PCR
Data Source
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AI summary
Provided herein, among other things, are a variety of methods for transposase-5 mediated tagging and amplification of short DNA fragments, e.g., between about 150 bp and 1.5 Kb in length. In some aspects, the method includes tagging the DNA fragments with a first primer sequence using barcoded transposases followed by a primer extension reaction to introduce a second primer sequence, e.g., using random or gene-specific primers. Kits for performing this method are also provided.