Digital PCR Quantification of NGS Library Members
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for next-generation sequencing (NGS) library quantification face challenges in standardizing library characterization due to varying adapter lengths and quality, leading to issues with malformed library members that consume resources and reduce sequencing efficiency.
Innovation Solution
A digital PCR method using short primers and probes targeting conserved adapter regions with locked nucleic acid analogues, allowing for precise quantification and identification of library members through digital amplification and fluorescence detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR quantification methods are used, then library concentration can be measured, but measurement precision is reduced due to varying adapter lengths and malformed library members
Solution Approach 1:
The invention segments the library population into distinct groups based on adapter configuration (full adapters, single adapter, no adapter) using digital PCR partitioning. Each partition independently detects and quantifies specific adapter types, enabling precise measurement of functional library members separate from malformed ones, thereby resolving the contradiction between measurement precision and reliability.
Solution Approach 2:
The invention introduces probe-based detection as an intermediary mechanism between the library samples and quantification readout. Fluorescent probes specific to adapter sequences mediate the detection process, allowing reliable distinction between functional and malformed library members even when conventional methods fail, thus improving both measurement precision and quantification reliability.
2Productivity
If library pooling is increased to reduce per-sample cost, then productivity increases, but measurement precision deteriorates due to widely varying library concentrations
Solution Approach 1:
The invention segments the quantification process to separately measure functional library members (with both adapters) from total DNA concentration. This enables accurate determination of effective library concentration in pooled samples, allowing researchers to optimize pooling strategies that maximize throughput while maintaining precise concentration measurements for proper normalization.
Solution Approach 2:
The invention changes the measurement parameter from total DNA concentration to functional library member concentration by using probe-based detection of specific adapter sequences. This parameter change enables precise quantification even in highly pooled samples with varying compositions, supporting increased productivity without sacrificing measurement precision.
3Length of moving object
If adapter regions are made shorter to reduce library size, then length decreases, but device complexity increases due to difficulty in assay development
Solution Approach 1:
The invention introduces probe-based detection as an intermediary that simplifies assay development for short adapters. The probes act as mediators that can be designed to specifically bind short adapter regions, making the detection process more reliable and less complex than developing short primer sets, thus resolving the contradiction between reduced adapter length and assay development complexity.
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 enables accurate and simultaneous quantification of different library types, reducing the impact of malformed members and improving sequencing efficiency by providing absolute quantification and binary values for library presence, thus enhancing the precision and accuracy of NGS library analysis.
Implementation Method 1
the upstream and downstream PCR primers in said PCR reaction bind in the upstream and downstream primer region of the amplification product
Implementation Method 2
amplifying said amplification product in a PCR reaction
Implementation Method 3
at least two primers having a length of between 10 and 22 nucleotides and optionally comprising one or more locked nucleic acid nucleotide analogues or another nucleotide analogue that increase template binding strength
Implementation Method 4
digital amplification and fluorescence detection
Data Source
AI summary
The invention relates to a method for examining a nucleic acid amplification product, comprising the steps of a. providing an amplification product stemming from a preferably exponential amplification comprising i. an upstream and downstream primer region as well as optionally and preferably, ii. an amplified target region between said upstream and downstream primer region, b. examining the amplification product by amplifying said amplification product in a PCR reaction, wherein i. the upstream and downstream PCR primers in said PCR reaction bind in the upstream and downstream primer region of the amplification product, ii. a first and a second oligonucleotide probe are placed downstream of the first and second PCR primers and within the upstream and the downstream primer region, c. performing a PCR reaction and examining the newly formed amplification product, wherein the upstream and downstream primer regions of step a.i.) have a length of under 35 nucleotides and over 20 nucleotides.