Troubleshooting guide for DDNS

Joyce Akello; Alex Shaw; Catherine Troman; Erika Bujaki; Manasi Majumdar; Aine OToole; c.ansley; Zoe Vance; rachel.colquhoun; Javier Martin; Nick Grassly; Andrew Rambaut

Aug 29, 2024

Version 2

Troubleshooting guide for DDNS V.2

DOI

dx.doi.org/10.17504/protocols.io.8epv5xe84g1b/v2

¹Imperial College London;
²Medicines and Healthcare products Regulatory Agency;
³University of Edinburgh

Poliovirus Sequencing Consortium

Joyce Akello

Imperial College London

DOI: dx.doi.org/10.17504/protocols.io.8epv5xe84g1b/v2

Document Citation: Joyce Akello, Alex Shaw, Catherine Troman, Erika Bujaki, Manasi Majumdar, Aine OToole, c.ansley, Zoe Vance, rachel.colquhoun, Javier Martin, Nick Grassly, Andrew Rambaut 2024. Troubleshooting guide for DDNS. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xe84g1b/v2Version created by Joyce Akello

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: October 12, 2023

Last Modified: August 29, 2024

Document Integer ID: 106664

Keywords: DDNS, Poliovirus direct detection, Nanopore sequencing, Troubleshooting DDNS

Funders Acknowledgement:

Bill and Melinda Gates Foundation

Abstract

This document provides general guidance for troubleshooting problems encountered in the DDNS method. It outlines the factors that might influence the outcome of the DDNS PCR assay run, the common problems encountered, possible causes of the problems, and provides recommendations on corrective action and preventative action. 
This document is primarily developed for DDNS performed on stool samples, but included are other factors to consider for other sample types.

DDNS troubleshooting guide

Purpose
To outline the most common sources of DDNS assay run failure / unacceptable results, reduced/lower sensitivity
than expected and provide recommendation on how to avoid these pitfalls and improve performance. 


Troubleshooting considerations.
Table1.  Factors to consider when troubleshooting DDNS results. 
AB
  Factor    Considerations  
 Nucleic acid (RNA) template•	Stool sample condition (timeline of collection and processing, transport, and storage conditions)
•	Quality (nucleic acid extraction method, correct performance of procedure for extraction, handling, and storage of RNA extract)
•	Purity (presence of PCR Inhibitors and /or contaminants affecting RT-PCR amplification)
•	Number of freeze-thaw cycles (maximum of three freeze-thaws only)
  PCR and
  sequencing reagents/ kit  •	Quality (Lot to lot variation because of manufacturing or transport and storage conditions)
•	Shelf life (expiration date, stability)
•	Integrity (storage and transport condition, packaging, number of freeze-thaw cycles)
•	Sequencing reagents compatible with the flow cell - device

Run controls (Positive control CVA20 and negative control Nuclease free water)  •	Quality (lot to lot variation as result of manufacturing or transport conditions)
•	Shelf life (expiration date)
•	Stability (storage temperature, reconstitution of the positive control CVA20, aliquoted for single use only for positive control CVA20)
•	Integrity (storage and transport condition, packaging)
 DDNS PCR
  assay programs  •	Thermal profiles properly programmed in the instrument with temperature ramping speed consideration
 Conventional end-point/ block-based PCR thermal cycler  •	Compatibility of PCR consumables used (PCR plates, strip tubes, sealing film, caps etc that may affect PCR amplification if not sealed properly)
•	Equipment maintenance and performance monitoring
Nanopore sequencing  •	Sequencing run settings
•	Sequencing reagent - flow cell - device  – software compatibility
Operator error (manual, automated, result interpretation error)  •	Procedure execution errors (consumables, protocol steps, volumes, device settings)
•	Analysis errors
Laboratory’s internal quality control  •	Standardized checkpoints to ensure proper execution and risks for failure in each step are minimised
•	Quality indicators in place to serve as tools for troubleshooting
DDNS Data analysis (using PiranhaGui
  Software)  •	Software issue (version, compatibility, input/ output access)
•	Sample/run data input error
General laboratory practice  •	Cleanliness of workspaces and equipment
•	Equipment and pipette calibration and maintenance 
•	Labelling of samples, reagents, reactions
Below is  a list of commonly encountered DDNS PCR assay run errors, possible causes, recommended corrective, and preventive actions. 

No amplification detected  / sequence obtained in samples previously identified as positive by culture or ITD-qPCR provided that positive and negative controls are valid
ABCD
Problematic resultPossible causeRecommended solution/ corrective actionRecommended preventive action
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are validErroneous RNA extraction procedure performedTo verify, repeat the DDNS PCR assay using the same RNA extract of the failed samples; If the result of the repeat test remains negative, repeat the RNA extraction method with the recommended kit•	Ensure trained personnel.
•	Prepare quick guides and extraction plate maps ready on hand 
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are validExtraction method used not recommended for DDNS method Re-extract the stool supernatant using the MagMAX Viral RNA Isolation kit manually or automated; If this extraction method is not available in your lab, use either the Qiagen QIAamp Viral RNA Mini kit or Roche High Pure Viral RNA kitEnsure that all the extraction method steps are followed according to  protocol
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are validPCR inhibitorsConfirm presence of inhibitors by spiking the positive control virus (CVA20) into the sample; if the positive control does not amplify, then inhibitors are suspected; if inhibitors are suspected, use BSA in the mastermix to help overcome PCR inhibitors; alternatively, if inhibitors are suspected, dilute existing stool supernatant or eluted RNA 10-fold  and repeat the RT-PCR. If a signal; is observed using the diluted sample, inhibitors are suspected•	Ensure that all the extraction method steps are followed adequately.
•	Use BSA in the RT-PCR mastermix to help overcome PCR inhibitors.
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are validSample mislabellingCheck sample labelling, with crosschecking EPID and Lab ID numbersEnsure correct sample labelling and recording
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are validPoor specimen quality or RNA template may be damaged/ degradedConfirm by repeating the test from nucleic acid extraction to PCR; if the issue persists, use a semi-nested PCR approach which has a shorter first round PCR product so likely to amplify a bit betterEnsure that collected samples are properly stored and processed.
Negative result for some samples: amplification detected / sequence obtained in some but
not all samples that tested positive by culture or ITD. Provided that positive and negative controls for all targets are valid.
ABCD
Problematic resultPossible causeRecommended solution/ corrective actionRecommended preventive action
Negative result for some samples: amplification detected / sequence obtained in some but not all samples that tested positive by culture or ITD. Provided that positive and negative controls for all targets are valid.Inconsistent pipetting technique in template additionRepeat the DDNS assay for the concerned samples using proper pipetting technique; merely repeating the sample may also lead to additional detection•	Ensure trained personnel.
•	Always check liquid volumes in pipette tips for each addition
•	Ensure pipettes are services and calibrated
Negative result for some samples: amplification detected / sequence obtained in some but not all samples that tested positive by culture or ITD. Provided that positive and negative controls for all targets are valid.RNA samples in a PCR plate/run were extracted using different methodologies with different performance characteristicsRe-extract and repeat the DDNS PCR assay on the concerned samples using the MagMAX Viral RNA isolation kit or QIAamp Viral RNA Mini Kit or Roche High Pure Viral RNA kit Ensure trained personnel
Negative result for some samples: amplification detected / sequence obtained in some but not all samples that tested positive by culture or ITD. Provided that positive and negative controls for all targets are valid.Failure to extract viral target RNA due to presence of virus below the detectable limit (LOD) of the DDNS assayPerform DDNS on culture isolates  of the stool supernatant  suspected to contain virus levels below the DDNS LOD 
Ensure trained personnel
Negative result for some samples: amplification detected / sequence obtained in some but not all samples that tested positive by culture or ITD. Provided that positive and negative controls for all targets are valid.Sample mislabellingCheck sample labelling, with crosschecking EPID and Lab ID numbersEnsure correct sample labelling and recording
Negative result for some samples: amplification detected / sequence obtained in some but not all samples that tested positive by culture or ITD. Provided that positive and negative controls for all targets are valid.PCR machine failure. Consider if PCR reagents/ kit performance  verified prior to use and PCR reagents/kit lot passed QC prior to use and all samples including run controls show no amplification •	Retest the whole plate using different PCR machine (previously verified to work with the assay)
•	Contact technical service engineer and equipment supplier for technical assistance
•	Ensure preventive maintenance plan for the equipment is strictly followed.

•	Only use PCR machines with valid calibration certificates

•	Ensure all users are trained on proper use of machines
No amplification detected/ sequence obtained in all samples including positive and negative controls
ABCD
Problematic resultPossible causeRecommended solution/ corrective actionRecommended preventive action
No amplification detected/ sequence obtained in all samples including positive and negative controlsExtraction Reagent/ PCR kit lot issue•	Check that the reagents/ PCR kit lot used has not expired. •	Verify this by parallel testing a reagent lot that has been previously confirmed to be working vs the reagent lot suspected to give problematic results.  Use positive and negative control for the parallel-test in duplicate. If the reactions using the reagent lot previously confirmed to be working provides the expected results and the reactions for the suspected problematic lot does not provide the expected results, then this confirms that a bad reagent lot was used; Retest the whole plate using new reagent lot of QC passed negative  template control material; Prepare a report and contact the supplier/ manufacturer regarding the issue and request for technical assistance•	Only use reagents/ PCR kits within date

•	Verify if reagents were transported and received according to manufacturer recommendations.
•	Check if reagents are stored according to manufacturer recommendations.
QC check new lots by running positive and negative controls using a lot verified to be working and the incoming reagent lot (old vs new)
No amplification detected/ sequence obtained in all samples including positive and negative controlsOne or more of the mastermix components  from either the PanEV (First round PCR) or VP1 PCR (second round PCR) are limiting the reaction due to missed addition, incorrect calculation, or expired reagents.•	Re-check calculation on worksheet

•	Repeat test using new stock reagents; if it is a case of a bad reagent lot caught in the lot-testing, then contact the manufacturer/supplier

•	Ensure that all reagents /Kits used are within expiry.
•	Record kit and reagent lot numbers on worksheet
•	Record/tick every addition of components while preparing mastermixes
•	Record/tick addition steps during protocol i.e. forward primer, barcoded primers, first PCR product 
No amplification detected/ sequence obtained in all samples including positive and negative controlsIncorrect PCR program or cycling conditions were usedRepeat amplification from RT stepRecord/tick each step in the protocol to ensure correct thermocycler programs and cycling conditions are used
No amplification detected/ sequence obtained in all samples including positive and negative controlsFailed RNA extraction•	Re-extract and repeat  the DDNS assay on all samples using the MagMAX Viral RNA isolation Kit or QIAamp Viral RNA Mini Kit or Roche High Pure Viral RNA kit.•	Ensure that all steps of the extraction protocol are followed adequately.
•	Control RNA extraction and first PCR separately by introducing a separate positive control for the first PCR in the form of a previously extracted RNA that successfully amplified before
•	Check that all recommendations and notes in the RNA extraction protocol are considered.
Identical sequences appearing at low read numbers (10-100) over multiple samples
ABCD
Problematic resultPossible causeRecommended solution/ corrective actionRecommended preventive action
Identical sequences appearing at low read numbers (10-100) over multiple samplesContamination of equipment due to repeated DDNS performance•	Perform a deep clean of workstations, pipettes and equipment with nucleic acid degradation solutions (e.g. DNAZap).•	Clean workstation and pipettes between runs                           •	Perform a routine deep clean every five DDNS runs                          •      Separate work areas for pre- and post-amplification steps
Amplification is detected but no sequences are produced
ABCD
Problematic resultPossible causeRecommended solution/ corrective actionRecommended preventive action
Amplification is detected but no sequences are producedIncorrect sequencing run settings used•	Make sure you have selected the correct sequencing and barcoding kits in the run options.
•	If pod5 files are being produced, you can re-call the data post-run. These files may be in pod5_fail, or pod5_skip
When setting up the run, ensure you select SQK-LSK114, and EXP-PBC096
Amplification is detected but no sequences are producedIncompatible kit reagents used•	If you have multiple ONT kits, it may be possible that you took the adapter mix from an incompatible kit for the final adapter ligation step.
•	You would need to repeat the library preparation, ensuring that you use the correct reagents from the SQK-LSK114 kit •	Keep ONT reagents in their original packaging and make sure you only take from that kit box when you are doing the library preparation.
Amplification is detected but no sequences are producedSamples missed during pooling•	If individual samples are accidentally missed during pooling, there will be no reads for that barcode.•	Take extra care to pool all barcoded samples and check volume in pipette tip for each addition.
For Issues during the sequencing run please see some suggested causes and solution on the Nanopore page here

MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell check
ABCD
Observed issuePossible causeRecommended solution/ corrective actionRecommended preventive action
MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell CheckAn air bubble was introduced into the nanopore array•     After the Flow cell check, it is important to remove any air bubbles near the priming before priming the flow cell. if not removed, the air bubble can travel to the nannopore array and cause irreversible damage to the nanopores             
  •	 Avoid introducing air as this will permanently damage the intehrity of the pore membranes"Use good practice pipetting to prevent introducing air into the center array which could have a detrimental effect on the membranes and subseqeunctly the active pore count.
MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell CheckThe flow cell is not correctly inserted into the deviceStop the seqeuncing run, remove the flow cell from the device and insert it again ensuring that the flow cell is firmly seated in the device and that it has reached the target temperature. if applicable, try a different position on the seqeuncing device (GridION/PromethION)Correctly insert the flow cell into the seqeuncing device, ensure that it is firmly seated in the device and that is has reached the target temperature
MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell CheckContaminants in the library have damaged the membranes or blocked the pores and therefore unable to seqeunce. This manifests itself as a build-up of "Unavailable" pores over time.If, despite the channel blocking, the library is still producing a sufficient number reads, carry on with the sequencing experiment.  Otherwise, stop the sequencing run in MinKNOW. Then wash out the library from the flow cell using the instructions for the Flow Cell Wash Kit.Then prepare another library and load it on the flow cell. Ensure to follow the DDNS protocol adequately to avoid  carry over of chemical components that could contaminate the RNA sample and subseqeunently have a significant effect on downstream library preparation efficiency, and therefore sequencing throughput.
MinKNOW  script failed.
ABCD
Observed issuePossible causeRecommended solution/ corrective actionRecommended preventive action
MinKNOW shows "Script failed"Restart the computer and then restart MinKNOW. if the issue persists, please collect the MinKNOW log files and contact technical support
Shorter than  expected read length 
ABCD
Observed issuePossible causeRecommended solution/ corrective actionRecommended preventive action
Shorter than expected read lengthUnwanted fragmentation of the DNA sample during library preparationDuring library preparation, avoid pipetting and vortexing when mixing reagents.Flick or invert the tube when mixing reagents during library preparation
Below is  a list of commonly encountered errors when installing and running PIRANHA GUI
Docker installation errors
ABCD
Error/Issue raisedPossible causeRecommended solution/ corrective actionRecommended preventive action
Docker installation errorsError pops up asking to update WSL2 (Windows Subsystem for Linux)If Docker is using WSL2 backend it may need updating, you can download the executable for doing this on the Microsoft website.Install this update before installing docker, but it is also ok to wait until it shows the error, in case you do not need to do this.
Docker installation errorsError pops up mentioning HyperV, HyperVisor, or virtualisation capabilities•	Go to “Turn Windows features on or off” and make sure HyperV is enabled, you may need to restart the computer after this.                          •	If you are not able to turn HyperV on and it says, “Virtualisation not enabled in firmware”, you need to start up your device in bios settings and enable virtualisation here before turning it on as above. Check virtualisation is enabled in Windows features before starting up Docker for the first time.
Docker failed to start error
ABCD
Error/Issue raisedPossible causeRecommended solution/ corrective actionRecommended preventive action
Docker failed to startHyper-V is not enabled or not installedEnable Hyper-V, by following the below steps:              
a) Open the Control Panel on your Windows system.    b) Click on “Programs and Features” and then click on “Turn Windows features on or off”.                      
c) Scroll down the list of features and look for the “Hyper-V” option. Check the box next to it to enable Hyper-V.      
d) Click on “OK” to save your changes.                 e) Restart your computer to apply the changes. Once Hyper-V is enabled, try starting Docker Desktop againEnsure that Hyper-V is enabled and installed
Docker failed to startVirtualization is not enabled in BIOSEnable virtualization in the BIOS. see the link here on how to enable virtualisation: https://collabnix.com/error-docker-failed-to-start-docker-desktop-for-windows/. Ensure that virtualisation in the BIOS is enabled
PIRANHA GUI says Docker is not installed when it is
ABCD
Error/Issue raisedPossible causeRecommended solution/ corrective actionRecommended preventive action
PIRANHA GUI says Docker is not installed when it isDocker is not openClose PIRANHA GUI, open Docker, then reopen PRIANHA GUIOpen Docker first before opening the PIRANHA GUI
PIRANHA GUI says Docker is not installed when it isDocker is not starting up properly or failed to startMake sure that Hyper-V is installed and enabled on your system to allow Docker to start properlyEnsure that Hyper-V is enabled and installed
You start analysis but it immediately stops with an error
ABCD
Error/Issue raisedPossible causeRecommended solution/ corrective actionRecommended preventive action
You start analysis but it immediately stops with an errorThe column headers for the barcode csv are incorrectMake sure the first two columns are labelled “barcode” and “sample” all lower case and with no accidental spaces addedDouble check the barcode csv file before running PIRANHA, and you can reuse csv files that have worked before rather than creating new each time. 
You start analysis but it immediately stops with an errorThe csv is saved as the wrong file typePIRANHA GUI is very sensitive to the different csv file types. Ensure that it is saved as a csv (comma delimited)If you have a csv that has worked previously, you can copy this to your desired location and reuse it, ensuring that the correct information for the current run is input. 
You run analysis but the output is empty or all 0 reads
ABCD
Error/Issue raisedPossible causeRecommended solution/ corrective actionRecommended preventive action
You run analysis but the output is empty or all 0 readsThe sample/barcode associations in the barcode csv are incorrectDouble check in the barcodes csv that you have put the correct barcodes to the correct samples. You can also check your fastq_pass files to double check the barcodes detected if you have doubtsAs soon as you have added barcodes to a sample, take note and log this in the barcodes csv
You run analysis but the output is empty or all 0 readsThe file path for the fastq_pass is incorrectGo back to the start page and check the location selected. Make sure you click all the way through to where the barcode directories are and not to a higher-level directory.Click through all the way to a barcode directory when selecting the fastq_pass folder in the GUI.
You run analysis but the output is empty or all 0 readsError during transfer of your fastq filesCheck inside your fastq_pass barcode directories to make sure they contain fastq files and the number of files match the number in the original fastq_pass folder created by MinKNOW.Make sure everything has finished copying before removing any external hard drives or USB sticks and eject properly before removing. 
You run analysis but the output is empty or all 0 readsThe GUI cannot access your fastq filesSometimes, due to read/write permissions, the GUI cannot access the fastq files you point it to. Try moving your files to another destination on your device and see if that helps. •	When transferring fastq files, put them into a directory within your documents rather than on the desktop or a shared drive. You can move the analysis output and fastq files elsewhere after running if you wish.
•	Check during the initial phase of the analysis to see how many fastq files were located by PIRANHA to see if they were accessed
You run analysis but the output is empty or all 0 readsIncorrect analysis settings were usedCheck the analysis settings in PIRANHA options as incorrect read length settings can lead results in 0 mapped readsIt is recommended to check the PIRANHA analysis option settings to ensure they are all appropriate for the actual analysis
Your controls have not been recognised in the run report 
ABCD
Error/Issue raisedPossible causeRecommended solution/ corrective actionRecommended preventive action
Your controls have not been recognised in the run reportYou have not named your controls to match the defaultPIRANHA looks for samples named “positive” and “negative” in the barcodes.csv. Rename your controls appropriately.Name your controls as positive or negative when you first fill in the details in the barcodes.csv
Your controls have not been recognised in the run reportRename your controls appropriately.•	You can tell PIRANAH GUI the names of your controls in the Run Options if you are not staying with the default.                     •	If what you write in does not match the name in the barcodes csv, PIRANHA will not be able to make the association. Ensure that if you specify control names that are different from the default that you match this correctly to the names in the barcodes.csv fileKeep to the default nomenclature of positive and negative

 

	A	B
	Factor	Considerations
	Nucleic acid (RNA) template	• Stool sample condition (timeline of collection and processing, transport, and storage conditions) • Quality (nucleic acid extraction method, correct performance of procedure for extraction, handling, and storage of RNA extract) • Purity (presence of PCR Inhibitors and /or contaminants affecting RT-PCR amplification) • Number of freeze-thaw cycles (maximum of three freeze-thaws only)
	PCR and sequencing reagents/ kit	• Quality (Lot to lot variation because of manufacturing or transport and storage conditions) • Shelf life (expiration date, stability) • Integrity (storage and transport condition, packaging, number of freeze-thaw cycles) • Sequencing reagents compatible with the flow cell - device
	Run controls (Positive control CVA20 and negative control Nuclease free water)	• Quality (lot to lot variation as result of manufacturing or transport conditions) • Shelf life (expiration date) • Stability (storage temperature, reconstitution of the positive control CVA20, aliquoted for single use only for positive control CVA20) • Integrity (storage and transport condition, packaging)
	DDNS PCR assay programs	• Thermal profiles properly programmed in the instrument with temperature ramping speed consideration
	Conventional end-point/ block-based PCR thermal cycler	• Compatibility of PCR consumables used (PCR plates, strip tubes, sealing film, caps etc that may affect PCR amplification if not sealed properly) • Equipment maintenance and performance monitoring
	Nanopore sequencing	• Sequencing run settings • Sequencing reagent - flow cell - device – software compatibility
	Operator error (manual, automated, result interpretation error)	• Procedure execution errors (consumables, protocol steps, volumes, device settings) • Analysis errors
	Laboratory’s internal quality control	• Standardized checkpoints to ensure proper execution and risks for failure in each step are minimised • Quality indicators in place to serve as tools for troubleshooting
	DDNS Data analysis (using PiranhaGui Software)	• Software issue (version, compatibility, input/ output access) • Sample/run data input error
	General laboratory practice	• Cleanliness of workspaces and equipment • Equipment and pipette calibration and maintenance • Labelling of samples, reagents, reactions

A	B	C	D
Problematic result	Possible cause	Recommended solution/ corrective action	Recommended preventive action
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are valid	Erroneous RNA extraction procedure performed	To verify, repeat the DDNS PCR assay using the same RNA extract of the failed samples; If the result of the repeat test remains negative, repeat the RNA extraction method with the recommended kit	• Ensure trained personnel. • Prepare quick guides and extraction plate maps ready on hand
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are valid	Extraction method used not recommended for DDNS method	Re-extract the stool supernatant using the MagMAX Viral RNA Isolation kit manually or automated; If this extraction method is not available in your lab, use either the Qiagen QIAamp Viral RNA Mini kit or Roche High Pure Viral RNA kit	Ensure that all the extraction method steps are followed according to protocol
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are valid	PCR inhibitors	Confirm presence of inhibitors by spiking the positive control virus (CVA20) into the sample; if the positive control does not amplify, then inhibitors are suspected; if inhibitors are suspected, use BSA in the mastermix to help overcome PCR inhibitors; alternatively, if inhibitors are suspected, dilute existing stool supernatant or eluted RNA 10-fold and repeat the RT-PCR. If a signal; is observed using the diluted sample, inhibitors are suspected	• Ensure that all the extraction method steps are followed adequately. • Use BSA in the RT-PCR mastermix to help overcome PCR inhibitors.
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are valid	Sample mislabelling	Check sample labelling, with crosschecking EPID and Lab ID numbers	Ensure correct sample labelling and recording
No amplification detected / seqeunce obtained in samples previosuly identified as positive by culture or ITD-qPCR provided that positive and negative controls are valid	Poor specimen quality or RNA template may be damaged/ degraded	Confirm by repeating the test from nucleic acid extraction to PCR; if the issue persists, use a semi-nested PCR approach which has a shorter first round PCR product so likely to amplify a bit better	Ensure that collected samples are properly stored and processed.

A	B	C	D
Observed issue	Possible cause	Recommended solution/ corrective action	Recommended preventive action
MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	An air bubble was introduced into the nanopore array	• After the Flow cell check, it is important to remove any air bubbles near the priming before priming the flow cell. if not removed, the air bubble can travel to the nannopore array and cause irreversible damage to the nanopores • Avoid introducing air as this will permanently damage the intehrity of the pore membranes"	Use good practice pipetting to prevent introducing air into the center array which could have a detrimental effect on the membranes and subseqeunctly the active pore count.
MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	The flow cell is not correctly inserted into the device	Stop the seqeuncing run, remove the flow cell from the device and insert it again ensuring that the flow cell is firmly seated in the device and that it has reached the target temperature. if applicable, try a different position on the seqeuncing device (GridION/PromethION)	Correctly insert the flow cell into the seqeuncing device, ensure that it is firmly seated in the device and that is has reached the target temperature
MinKNOW reports a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	Contaminants in the library have damaged the membranes or blocked the pores and therefore unable to seqeunce. This manifests itself as a build-up of "Unavailable" pores over time.	If, despite the channel blocking, the library is still producing a sufficient number reads, carry on with the sequencing experiment. Otherwise, stop the sequencing run in MinKNOW. Then wash out the library from the flow cell using the instructions for the Flow Cell Wash Kit.Then prepare another library and load it on the flow cell.	Ensure to follow the DDNS protocol adequately to avoid carry over of chemical components that could contaminate the RNA sample and subseqeunently have a significant effect on downstream library preparation efficiency, and therefore sequencing throughput.

A	B	C	D
Error/Issue raised	Possible cause	Recommended solution/ corrective action	Recommended preventive action
Docker installation errors	Error pops up asking to update WSL2 (Windows Subsystem for Linux)	If Docker is using WSL2 backend it may need updating, you can download the executable for doing this on the Microsoft website.	Install this update before installing docker, but it is also ok to wait until it shows the error, in case you do not need to do this.
Docker installation errors	Error pops up mentioning HyperV, HyperVisor, or virtualisation capabilities	• Go to “Turn Windows features on or off” and make sure HyperV is enabled, you may need to restart the computer after this. • If you are not able to turn HyperV on and it says, “Virtualisation not enabled in firmware”, you need to start up your device in bios settings and enable virtualisation here before turning it on as above.	Check virtualisation is enabled in Windows features before starting up Docker for the first time.

Public workspaceTroubleshooting guide for DDNS V.2

Troubleshooting guide for DDNS V.2