Molecular Tag Attachment for False Positive Detection in NGS
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Solution Overview
Problem
Current next-generation sequencing (NGS) techniques struggle to distinguish true low-frequency variants from false positives caused by errors during sample preparation or sequencing, such as DNA modifications like oxidation of cytosine, which can lead to incorrect SNP detection, particularly in liquid biopsy samples where disease cells are present in small proportions.
Innovation Solution
The method involves preparing double-stranded DNA fragments linked with oligonucleotide adaptors, denaturing them to generate separate strands, and using probes and primers with tag sequences to differentiate true positive from false positive modifications by sequencing and comparing the results from both strands, thereby identifying modifications that result in false positive SNP calls.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard NGS sequencing is performed on DNA fragments, then rapid and cost-effective analysis is achieved, but false positive SNP detections occur due to DNA modifications during sample preparation
Solution Approach 1:
A tag sequence is introduced as an intermediary element that is attached to the DNA fragment during library preparation. This tag serves as a mediator that links the original DNA fragment identity to its sequencing reads, enabling the system to track and compare sequences across different strands without altering the core sequencing process or requiring complex additional instrumentation.
Solution Approach 2:
The DNA analysis process is segmented into distinct components: the original DNA fragment, the tag sequence attached to it, and the sequencing reads generated. By segmenting the identification process this way, the patent enables independent verification of each strand's sequence against its tag, allowing false positives to be identified when there's inconsistency between strands while maintaining high throughput sequencing.
2Ease of manufacture
If DNA fragments undergo chemical modifications during isolation and processing, then sample preparation is completed, but the DNA polymerase cannot recognize original bases leading to false SNP calls
Solution Approach 1:
The tag sequence is attached to the DNA fragment during the library preparation process, before the DNA undergoes chemical modifications during isolation, fixation, and storage. This preliminary action ensures that the tag remains intact and can be used later to verify the original sequence context, even when the DNA bases themselves are modified and misread by polymerases.
3Measurement precision
If complementary tags are attached to every DNA fragment to identify modifications, then false positives can be distinguished, but the complexity and cost of library preparation increases
Solution Approach 1:
Instead of requiring complex complementary tags on every DNA fragment, the patent uses a simpler approach where a single tag sequence is attached during standard library preparation. The tag is designed to be sufficient for identification purposes without requiring elaborate complementary structures, thus achieving adequate precision with reduced complexity and cost.
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 effectively distinguishes true positive from false positive single nucleotide polymorphisms (SNPs) by leveraging the tag sequences to identify modifications that are consistent across both DNA strands, reducing the need for complementary tags on every DNA fragment and improving the accuracy of low-frequency event detection.
Implementation Method 1
A first probe may be annealed to the first strand DNA fragment. The first probe may include a probe oligonucleotide sequence that is complementary to and hybridizes to a first probe target region
Implementation Method 2
The first probe may be extended with a DNA polymerase using the first strand DNA fragment as a template, thereby generating a probe extension sequence
Implementation Method 3
The double-stranded DNA fragment may be denatured to generate a first strand DNA fragment and a second strand DNA fragment
Data Source
AI summary
Described herein are methods, compositions and kits for identifying modifications that could lead to false positive detections in nucleic acid sequencing. In some embodiments, the methods, compositions and kits provided herein are useful for reducing potential of false positive detection of variants caused by errors during sample preparation or sequencing.