May 18, 2023

Public workspaceQuality guidance on the use of run controls for direct detection of poliovirus by nanopore sequencing

DOI
dx.doi.org/10.17504/protocols.io.14egn2jdmg5d/v1
  • 1Imperial College London;
  • 2National Institute for Biological Standards and Control
Joyce Akello
Open access
DOI: dx.doi.org/10.17504/protocols.io.14egn2jdmg5d/v1
Document Citation: Joyce Akello, Manasi Majumdar, Alex Shaw, Catherine Troman, Erika Bujaki, Javier Martin, Nick Grassly 2023. Quality guidance on the use of run controls for direct detection of poliovirus by nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2jdmg5d/v1
License: This is an open access document distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: February 27, 2023
Last Modified: April 19, 2024
Document Integer ID: 77699
Keywords: Poliovirus, Direct detection, Nanopore sequencing, Extraction run controls
Funders Acknowledgement:
Bill and Melinda Gates Foundation
Abstract
This document describes the laboratory process for the use of run controls (positive and negative controls) for performing direct detection of poliovirus by nanopore sequencing (DDNS assay).

Guidance on the use of run controls for direct detection of poliovirus by nanopore sequencing

Purpose
To outline the key steps of how and when to use the run controls for DDNS assay including preparation, storage, and outcome interpretation. The run controls are used to demonstrate that the entire DDNS workflow starting from RNA extraction to obtaining a sequence is successfully performed.

Associated forms & documents

Reagents
  • Coxsackie virus A20 –(Supplied by NIBSC, contact Dr. Manasi Majumdar at Manasi.Majumdar@nibsc.org and copy Dr. Javier Martin at Javier.Martin@nibsc.org)
  • Nuclease free water (NFW)
  • RNA extraction reagents (Recommend using the MagMAX Viral RNA Isolation kit performed manual or automated [ThermoFisher], the QIAamp Viral RNA Kit [Qiagen], or Roche High Pure Viral RNA Kit with proteinase K [Roche] )

Equipments
  • Microbiological Safety Cabinet, Class II (MSCII)
  • PPE (Gloves, Lab coat)
  • Vortex mixer
  • Benchtop centrifuge
  • Sterile 1.5ml Eppendorf tubes DNase/RNase free
  • Calibrated pipettes single channel (0.2µl to1000µl)
  • Sterile pipette tips with filters (10-1000µl)
  • Trend monitored freezer, -200C and refrigerator, 4 0C
  • Disposal Dispo-safe “sweetie” jar or bio-bin

Procedure
The positive and negative controls will both undergo all steps from sample RNA extraction, PCR amplification to nanopore sequencingrun in parallel with the samples. This is to ensurethat the entire DDNS workflow is controlled for cross contamination, failed RNA extraction, PCR amplification, and sequencing.


Run controls preparation, storage, and use
CVA20 positive control reconstitution


1. Working in the MSCII, reconstitute the vial containing the lyophilised CVA20 (freeze dried material) by adding 1mL of nuclease free water (NFW)

2. Vortex briefly to ensure that the material completely dissolves in water giving a colourless liquid

3. Aliquot the solution into single-use volumes of 30 µl in 1.5mL sterile eppendorf tubes (DNase/RNase free) and store all aliquots at -200C for future use.


Use of the positive extraction control (CVA20) and negative extraction control during RNA extraction.


4. Prepare the CVA20 positive extraction control for RNA extraction as follows:
a. Retrieve a 30 µl aliquot of the CVA20 from the -200C freezer
b. Allow to thaw at room temperature whilst in the MSCII cabinet
c. Briefly centrifuge for 5 secs.
d. Add 270 µl of NFW and pipette up and down to mix the solution. The solution is now ready for immediate RNA extraction



5. Prepare the negative extraction control for RNA extraction as follows:
a. Aliquot 300 µl of nuclease free water into a sterile 1.5mlEppendorf tube (DNase/RNase free)labelled ExNTC (extraction negative control)

6. Perform RNA extraction of the samples in parallel with the positive and negative control ensuring to include run extraction controls on the first and last RNA extraction batch run of the day

7.The elutes / purified RNA from the controls and samples following RNA extraction can now be processed according to the DDNS protocol.

Run controls validation
The PCR products of the run controls can be checked prior to sequencingto ensure that the RNA extraction and PCR amplification steps worked efficiently. Checking of the run control PCR products can be performed using the Agilent TapeStation system or gel electrophoresis. For the sample RNA extraction and the PCR amplification assay run to be accepted as valid, the PanEV RT-PCR and nested VP1 PCR results for the positive and negative extraction controls as well as the PCR no template control must be of the excepted results as resolved and visualised by the TapeStation or gel electrophoresis (see figure 1 below)

Figure 1. The Tapestation results for the DDNS assay controls from PanEV RT-PCR and nested VP1 PCR products (Panel A) and the gel electrophoresis results for the DDNS assay controls from PanEV RT-PCR and nested VP1 PCR products (Panel B). The positive extraction control labelled as CVA20 with a PanEV RT-PCR band at approximately 4.2kb and nested VP1 PCR band at approximately 1.2kb. The negative extraction control as ExNTC with no band in both the PanEV RT-PCR and nested VP1 PCR. The PCR no template control as PCR NTC with no band in both the PanEV RT-PCR and nested VP1 PCR.