VERA Sample Preparation for Viral Contaminant Identification
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Solution Overview
Problem
Existing Next-Generation Sequencing (NGS) technologies face challenges in efficiently detecting and identifying viral contamination in cell cultures due to complex viral genomes and RNA viruses, complicating library preparation and requiring optimized sample preparation strategies and automated workflows.
Innovation Solution
A streamlined sample preparation process, VERA (Viral Enrichment by Reducing Artifacts), which includes lysing eukaryotic cells, removing cellular debris, concentrating viral nucleic acids, and enzymatically digesting host nucleic acids using Benzonase®, OmniCleave™, and RiboShredder™, followed by real-time nanopore sequencing without amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If NGS-based approaches are used for broad testing of all viral genomes, then detection coverage is improved, but workflow complexity increases
Solution Approach 1:
The workflow is segmented into distinct modular steps: cell lysis, debris removal via filtration, concentration, nuclease treatment, and library preparation. Each module performs a specific function and can be independently optimized or automated, reducing overall workflow complexity while maintaining broad viral detection coverage
Solution Approach 2:
The methodology employs universal primers and nucleases that can detect and process diverse viral genomes (RNA and DNA, single-stranded and double-stranded) through a single standardized workflow, eliminating the need for virus-specific protocols and simplifying the overall process
2Adaptability or versatility
If RNA viruses are included in the testing, then detection versatility is improved, but library preparation difficulty increases
Solution Approach 1:
Reverse transcriptase is introduced as an intermediary enzyme that converts RNA viral genomes into cDNA, creating a universal intermediate form that can then be processed through the same library preparation workflow as DNA viruses. This mediator enables RNA virus detection without requiring separate complex protocols
Solution Approach 2:
The methodology incorporates temperature-controlled enzymatic reactions and optimized buffer conditions that accommodate both RNA and DNA processing requirements, changing physical and chemical parameters to enable universal library preparation across different virus types
3Measurement precision
If host genomic material is reduced, then viral signal-to-noise ratio is improved, but sample preparation time increases
Solution Approach 1:
Host genomic material is removed in advance through nuclease treatment before library preparation begins. By performing this degradation step preliminarily, the actual sequencing library preparation can proceed more efficiently with less host DNA interference, reducing the total time required for the critical downstream steps
Solution Approach 2:
Physical separation methods are replaced with enzymatic degradation using nucleases to remove host nucleic acids. This chemical/biological approach is more selective and efficient than mechanical methods, rapidly degrading host material while preserving viral genomes within protein capsids, thereby improving signal-to-noise ratio without significant time penalty
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 method enables rapid identification of potential viral contamination within one workday by enriching viral genomes, achieving viral reads of at least 51% of total reads, reducing the time and complexity of viral detection in cell cultures.
Implementation Method 1
The retentate is then treated with nucleases to digest eukaryotic nucleic acids
Implementation Method 2
Because viral particles protect viral nucleic acids, the viral nucleic acids are not digested by the nuclease treatment
Implementation Method 3
The viral nucleic acids are sequenced, for example by real time nanopore sequencing
Data Source
AI summary
The present disclosure is directed to a streamlined sample preparation process, VERA (Viral Enrichment by Reducing Artifacts), to tilt total genomic material in favor of DNA/RNA viral genomes. This reduction of host genomic artifacts can be completed in <8 hours from sample acquisition. Using a rapid library preparation protocol (˜1.5 minutes) and real-time nanopore sequencing, potential viral contamination, for example RNA viral contamination, can be identified in less than one workday from sample acquisition.