Sequencing Spike-In Controls for Contamination Detection and Quantitation
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Solution Overview
Problem
Current sequencing and analytical chemistry methods face challenges in detecting sample swapping and cross-contamination, and in accurately quantifying nucleic acids due to biases from DNA extraction and library preparation processes, which can be influenced by different microbial membranes and polymerase preferences.
Innovation Solution
The use of nucleic acid constructs with barcode and universal sequence fragments, encapsulated in simulated cell membranes or embedded in organism genomes, as spike-in controls to detect cross-contamination and sample swapping, and to create standard curves for quantitation, controlling for GC content and lysis efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA extraction and library preparation methods are used for sequencing analysis, then nucleic acid quantitation and sequencing can be performed, but biases are introduced due to different lysis efficiencies for different microbial membranes and polymerase preferences for certain GC content fragments
Solution Approach 1:
The patent introduces spike-in control nucleic acid constructs as intermediary reference materials that are added to samples before DNA extraction and library preparation. These controls contain known concentrations of nucleic acids with diverse GC content and are encapsulated in simulated cell membranes with varying lysis resistance. By comparing the recovery of these spike-in controls against their known input amounts, the method identifies and corrects for biases introduced by extraction and amplification processes, thereby enabling accurate quantitation of target nucleic acids despite the biases inherent in the sequencing workflow.
Solution Approach 2:
The patent systematically varies multiple parameters of the spike-in control constructs including GC content (ranging from low to high), simulated cell membrane lysis resistance (Gram-positive, Gram-negative, fungal-like), and nucleic acid concentration. By creating a panel of controls with different parameter combinations, the method can identify and correct for multiple sources of bias simultaneously, allowing accurate quantitation across diverse sample types and experimental conditions.
2Reliability
If standard sequencing controls are used to monitor sample integrity, then sample swapping and cross-contamination can be detected, but accurate quantitation of nucleic acids cannot be achieved due to workflow-induced biases
Solution Approach 1:
The patent designs spike-in control nucleic acid constructs that simultaneously serve multiple functions: (1) they contain unique barcodes or identifiers that enable detection of sample swapping and cross-contamination, and (2) they include known concentration information and diverse GC content that enable accurate quantitation of target nucleic acids. By integrating these multiple functions into a single control system, the patent eliminates the need for separate control mechanisms and resolves the contradiction between monitoring reliability and achieving measurement precision.
3Device complexity
If conventional quantitation methods are used without spike-in controls, then the sequencing process is simpler, but accurate quantitation of nucleic acids is compromised due to biases from DNA extraction and library preparation
Solution Approach 1:
The patent implements preliminary action by adding spike-in control nucleic acids to samples at the very beginning of the workflow, before DNA extraction and library preparation. This early incorporation ensures that the controls experience the same biases as the target nucleic acids throughout the entire process. By establishing this reference point upfront, the method enables accurate quantitation without requiring complex mid-workflow interventions or post-processing corrections, thus maintaining relative simplicity while achieving high precision.
Data Source
AI summary
The invention relates to control compositions for sequencing and for chemical analyses, such as analytical chemistry analyses. More particularly, the invention relates to control compositions for sequencing and for chemical analyses having at least one barcode sequence fragment and at least one universal sequence fragment, and to methods of their use.