Researchers from Ceres Nanosciences, USA, have presented a workflow for enrichment of nanoparticles to capture and concentrate particles of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from diagnostic and engineered samples.
This approach improves both column and magnetic bead-based RNA extraction protocols, providing significant improvements in sequencing results on the Oxford Nanopore (ONT) technologies Minion sequencing platform. Sequencing outputs showed significantly more mapped viral readings and 20-80% improved sequence coverage from diagnostic samples with low virus titers.
Study: Nanotrap particles improve nanopore sequencing of SARS-CoV-2 and other respiratory viruses. Image credit: Unitone Vector / Shutterstock
The team also demonstrated the same approach to enrich two other respiratory viruses, namely influenza A virus and respiratory syncytial virus, and highlighted its potential to improve real-time PCR and sequencing results for respiratory viruses other than SARS-CoV-2.
Background
Continuous genetic development of SARS-CoV-2 highlights the need to constantly identify and monitor the emergence of new viral variants and thus specific requirements for rapidly implementable accurate sequencing technologies.
This is where the ONT MinION sequencer comes into focus. As it is a portable and relatively inexpensive detection strategy, it can be used for rapid on-site detection and characterization of viral variants in the field. However, the utility of these portable sequencing tools is limited by the problems of base-calling accuracy that occur in samples with insufficient nucleic acid concentration.
The team from Ceres Nanosciences, in their recent research published on the preprint server bioRxiv *, has addressed this limitation by using an affinity-based magnetic hydrogel particle enrichment technology (Nanotrap particle enrichment technology) that can concentrate viral particles, leading to improved detection of many viral agents, including SARS-CoV-2. With superior base-calling, bioinformatics tools can reliably distinguish the genetic mutation in the variant from machine failure.
What did the researchers do?
The team developed three workflows to demonstrate the robustness and ease of use of Nanotrap particle technology: a manual method with a column-based RNA extraction (Nanotrap particle workflow 1), a manual method with a magnetic bead-based RNA extraction (Nanotrap particle workflow 2), and an automated method with a magnetic bead-based RNA extraction (Nanotrap particle workflow 3).
These workflows were performed in SARS-CoV-2 constructed (added 1:10 serial dilutions of heat-inactivated SARS-CoV-2 starting from 10.6 TCID50 / ml to 102 TCID50 / mL) viral transport media (VTM) and remaining diagnostic VTM samples. The nanotrap particle workflows (+ NT) were compared to the workflows without any Nanotrap particles (-NT).
Extracted RNA was prepared for sequencing using the ARTIC nCoV-2019 sequencing protocol and sequenced on the ONT Mk1C sequencing platform followed by data analysis.
In addition, real-time RT-PCR was performed on RNA extracted from all three workflows to identify a potential correlation between the two assays and confirm the presence of SARS-CoV-2.
Nanotrap particles improve sequencing results
With Nanotrap Workflow 1, Nanotrap particles significantly improved sequencing results at different virus concentrations compared to the workflow without Nanotrap particles. A 6.0X increase in readings mapped to SARS-CoV-2 was observed at 106 TCID50 / mL and a 2.0-fold increase was seen at 105 TCID50 / ml. These improvements were statistically significant. Viral detection in real-time qPCR was also significantly enhanced with two PCR Cycle Thresholds (Ct) across the first four serial dilutions.
Since column-based RNA extractions are more appropriate when working with a smaller number of samples, the team tried to assess the ability of the Nanotrap particles to improve sequencing using RNA extracted via magnetic beads (Nanotrap Workflow 2). Here, a 1.9X improvement in SARS-CoV-2 mapped readings was observed at 106 TCID50 / mL and an improvement of 1.4X at 105 TCID50/ ml. In addition, real-time qPCR detection of SARS-CoV-2 was improved, giving an average improvement of 1.5 Ct at 106-102 TCID50 / ml.
“RT-PCR results showed that Nanotrap particle enrichment was effective for lower concentration samples for both workflows, potentially suggesting that on an alternative sequencing platform with greater overall sensitivity, Nanotrap particles can also improve the sequencing of these virus samples by lower titer “, the team highlights.
To test the efficacy of Nanotrap particles with real clinical samples, the team evaluated the Nanotrap particle procedures using 10 diagnostic residues of clinical inoculation samples with reported qPCR Ct values from 24 to 35. The team quantified both total viral mapped readings and the corresponding percentage genome. coverage at 30x the depth of the treated samples.
Nanotrap Particle Workflow 1 resulted in a significant 7X improvement in the total viral load mapped across all 10 diagnostic residual samples, further resulting in an average increase in viral genome coverage of 52% over samples treated without Nanotrap particles. RT-PCR confirmed the presence of SARS-CoV-2 with a mean improvement of 4 Ct over no Nanotrap particle samples.
Automated Nanotrap Particle Workflow 3 method on automated KingFisher Apex System also showed improved results with an average improvement of 42X in total viral mapped readings, corresponding to an average 51% increase in viral genome coverage compared to (-NT) samples. Again, qPCR confirmed the presence of SARS-CoV-2 for all 10 samples with a mean improvement of 3.7 Ct.
Figure 1: Nanotrap particle workflow 1 improves sequencing of diagnostic residue SARS-CoV-2 samples. 10 SARS-CoV-2 positive diagnostic residues were treated using Nanotrap Particle Workflow 1 [+NT] or the RNEasy kit alone [-NT]. Samples were then sequenced on an ONT MinION R.9 flow cell and analyzed by Viral Mapped Reads for SARS-CoV-2 (A), Viral Genome Coverage at 30x depth (B) or RT-PCR (C). [+NT] was compared with [-NT] by paired t-test to assess the significance of increased viral detection (D), (E). *** p <0.001.
Improved detection of other respiratory viruses
To confirm the enriching ability of the nanotrap particles to other viruses and not just SARS-CoV-2, the team similarly tested RNA extraction with workflow 1 on engineered VTM samples supplemented with influenza A (H1N1) virus and RSV. It is noteworthy that Nanotrap particles were able to improve sequencing results for both viruses. As with SARS-CoV-2, the Nanotrap particles virally mapped readings increased by 4X for both influenza A and RSV compared to the samples treated without Nanotrap particles. In addition, Nanotrap particles significantly improved the detection of viral RNA by qPCR. Based on this experiment, the team suggests that Nanotrap particles can be used to identify and improve sequencing results of multiple viruses in VTM samples.
The preparation protocol of the sequencing library involves the use of various enzymes that may be adversely affected by the presence of harmful biological material present in the true human-derived diagnostic samples. Pretreatment with Nanotrap particles enables the capture of viral material, while reducing host cell debris and other contaminating material. Thus, the team suggests that workflows without Nanotrap particles are more likely to be affected by inhibition.
Conclusions
As viral genome coverage was greatly increased and almost improved by 80% in most diagnostic tests, the team suggests that the use of Nanotrap particles may allow for the accommodation of multiple clinical samples in a single run.
The team emphasizes the need for a simple and fast workflow to achieve a high sample flow in a public health environment and therefore recommends the automated Nanotrap Workflow 3 for such settings.
It is worth noting that this automated method would allow the processing of 96 samples in 1 hour, which is significantly faster and far more user-friendly than the manual column extraction method, making this an attractive proposition for medium to high flow laboratories.
In addition, Nanotrap particle workflows can be used to enhance broad-scale viral detection by sequencing as shown by sequencing experiments performed on influenza A and RSV.
Overall, the observations suggest that Nanotrap particle enrichment allows for sequencing of lower titer clinical specimens in VTM using the ONT MinION Sequencer, which may not otherwise have been suitable for sequencing.
A nanotrap particle concentration method paired with an ONT sequencing platform allows for a powerful sequencing tool that could potentially be implemented in an area with no access to more traditional sequencing or sample processing equipment, ”the team concludes.
Figure two: Nanotrap particle workflow 3 improves sequencing output of diagnostic SARS-CoV-2 samples. 10 SARS-CoV-2 positive diagnostic residues were treated using Nanotrap Particle Workflow 3 [+NT] or the MagMAX kit alone [-NT]. Samples were then sequenced on an ONT MinION R.9 flow cell and analyzed by Viral Mapped Reads for SARS-CoV-2 (A), Viral Genome Coverage at 30x depth (B) or RT-PCR (C). [+NT] was compared with [-NT] by paired t-test to assess the significance of increased viral detection (D), (E). * p <0.05, ** p <0.01.
*Important message
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered as crucial, guide clinical practice / health-related behavior or be treated as established information.
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