Asymmetric Adapter DNA Libraries for Absolute Retroviral Integration Detection
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Solution Overview
Problem
Current methods for detecting retroviral integration sites in gene and cell therapy are limited by low sensitivity and inability to achieve absolute quantification, particularly when integration frequencies are low, leading to inconsistent library quality and difficulty in establishing stable background filtering thresholds.
Innovation Solution
A method involving DNA library preparation through genomic DNA fragmentation, end-repair, A-tailing, and ligation with asymmetric double-strand adapters, followed by multiple rounds of PCR using internal reference genes and specific primers, and bioinformatics analysis to ensure consistent sequencing depth and accurate quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional DNA library preparation methods are used for detecting retroviral integration sites, then the detection process can be completed, but the sensitivity is low and absolute quantification cannot be achieved
Solution Approach 1:
The patent performs preliminary enrichment of retroviral integration sites through targeted PCR amplification using LTR-specific primers before library construction. This preliminary action concentrates the rare integration site sequences from the complex genomic DNA background, enabling subsequent high-sensitivity detection and absolute quantification that would be impossible with conventional direct sequencing approaches
Solution Approach 2:
The patent introduces an intermediary enrichment step using LTR-specific primers and adapter sequences that bridge the genomic DNA and sequencing library. This intermediary approach allows selective amplification and quantification of integration sites while maintaining the ability to perform absolute quantification through controlled PCR cycles and internal reference genes
2Measurement precision
If multiple rounds of PCR amplification are performed to enrich integration sites, then detection sensitivity improves, but amplification errors increase
Solution Approach 1:
The patent performs multiple rounds of PCR amplification (first round with LTR-specific primers, second round with adapter-specific primers) in continuous sequence without intermediate purification that would lose material. This continuous amplification maintains sensitivity while using high-fidelity polymerases to minimize error accumulation across rounds
Solution Approach 2:
The patent optimizes PCR parameters including cycle number, annealing temperature, and polymerase selection to balance amplification efficiency and fidelity. By carefully controlling these parameters, the method achieves sufficient enrichment for sensitive detection while maintaining amplification accuracy through optimized reaction conditions
3Manufacturing precision
If conventional library preparation methods are used, then the process is simpler, but library quality is inconsistent and background filtering thresholds cannot be established
Solution Approach 1:
The patent incorporates internal reference genes with known copy numbers that serve as feedback controls throughout the library preparation and sequencing process. By comparing integration site reads to reference gene reads, the method establishes quality metrics and background filtering thresholds that ensure consistent library quality across different preparations
Solution Approach 2:
The patent segments the library preparation into distinct modular steps: enrichment PCR, adapter ligation, size selection, and amplification. Each module can be optimized and quality-controlled independently, improving overall library consistency while making the complex process more manageable and reproducible
Data Source
AI summary
The present disclosure belongs to the field of molecular biology, particularly to the technical field of gene analysis and detection, and specifically relates to a method for preparing a DNA library and a method for detecting a retroviral integration site. Specifically, the method for preparing the DNA library comprises the following steps: 1) fragmenting a genomic DNA from a retrovirus-infected cell to obtain DNA fragments; 2) subjecting the DNA fragments to end-repair, A-tailing, and ligation with an adapter to obtain a ligation product, wherein the adapter is an asymmetric double-strand adapter comprising a long-strand sequence and a short-strand sequence, wherein the long-strand sequence sequentially comprises, from a 5′ end to a 3′ end, a fixed sequence, a random UMI sequence, and an amplification primer binding sequence, and the short-strand sequence comprises a sequence complementary to the fixed sequence; and other steps. The detection method of the present disclosure has extremely high sensitivity, thus having good application potential.