Index Hopping Reduction in NGS via Primer Extraction
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Solution Overview
Problem
Next generation sequencing (NGS) platforms face challenges with index hopping, where barcode sequences incorrectly attach to template nucleic acids, leading to false-positive or false-negative results due to the presence of free and buried primers, which are not effectively addressed by existing technologies.
Innovation Solution
The method involves reducing the concentration of extendable free and buried primers relative to indexed sequencing templates through processes such as high-pressure liquid chromatography (HPLC), using terminal deoxytransferase and dideoxynucleotide triphosphates, scavenger nucleic acid molecules, or killer oligonucleotides and ligase to neutralize or remove these primers, thereby reducing index hopping.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplexed NGS sequencing is performed on combined patient samples, then sequencing efficiency is improved, but index hopping occurs leading to false-positive or false-negative results
Solution Approach 1:
The patent extracts and removes free primers from the sequencing sample through purification steps performed after amplification but before sequencing. This eliminates the source of index hopping while preserving the amplified products, thereby maintaining sequencing efficiency while improving result accuracy.
Solution Approach 2:
The patent performs preliminary purification to remove free primers before the sequencing step. By addressing the index hopping problem in advance, before the actual sequencing occurs, the method prevents false associations without compromising the ability to perform multiplexed sequencing efficiently.
2Productivity
If free primers are not removed from indexed samples, then the sequencing process is simpler and faster, but index hopping increases causing incorrect barcode associations
Solution Approach 1:
The patent specifically extracts free primers from the sample through purification methods. This removal step eliminates the harmful free primers that cause index hopping, thereby improving barcode association accuracy while maintaining a streamlined workflow that does not significantly impact sequencing speed.
Solution Approach 2:
The patent introduces a purification step as an intermediary process between amplification and sequencing. This intermediate step selectively removes free primers while preserving amplified products, acting as a mediator that resolves the conflict between speed and accuracy by preparing the sample in an optimal state for sequencing.
3Device complexity
If conventional amplification methods are used without primer removal, then the workflow is simpler, but buried primers cause index hopping artifacts
Solution Approach 1:
The patent extracts both free and buried primers from the sample through purification steps. This removal eliminates the source of index hopping artifacts while adding a manageable purification step to the workflow, thereby reducing harmful effects with acceptable increase in complexity.
Solution Approach 2:
The patent converts the harmful presence of free and buried primers into a beneficial purification opportunity. By deliberately introducing purification steps, the method transforms what was previously an overlooked problem into a controlled process step that actively eliminates index hopping artifacts while producing cleaner sequencing samples.
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 significantly reduces index hopping, enhancing the accuracy of NGS assays by minimizing incorrect barcode associations, leading to more reliable sequencing results.
Implementation Method 1
The method involves reducing the concentration of extendable free and buried primers relative to indexed sequencing templates through processes such as high-pressure liquid chromatography (HPLC)
Implementation Method 2
using terminal deoxynucleotidyl transferase and dideoxynucleotide triphosphates, scavenger nucleic acid molecules, or killer oligonucleotides and ligase to neutralize or remove these primers
Implementation Method 3
using terminal deoxynucleotidyl transferase and dideoxynucleotide triphosphates, scavenger nucleic acid molecules, or killer oligonucleotides and ligase to neutralize or remove these primers
Implementation Method 4
using terminal deoxynucleotidyl transferase and dideoxynucleotide triphosphates, scavenger nucleic acid molecules, or killer oligonucleotides and ligase to neutralize or remove these primers
Data Source
AI summary
The present disclosure relates to compositions and methods for reducing the concentration of extendable free and buried primers relative to amplification product in a sample. The disclosed methods and compositions can be used to reduce or eliminate index hopping in a next generation sequencing (NGS) platform.