Frequently Asked Questions
Your guide for an smooth onboarding and to help you and your team quickly and efficiently get familiar with the standard operating procedures (SOPs) of our wet lab.
FAQ Wet Lab
DTT is not on the material list?
Superscript buffer and DTT are included In the box of the superscript IV enzyme.
Ampure XP beads are not on the material list?
These are included in the ONT sequencing kit.
For what is the Vivaclear mini clarifying filter used?
Serum and CSF
- Transfer 500 μL into an Eppendorf centrifugal filter (0.8 μm) and centrifuge for 5 minutes at 2000 x g.
- Proceed with 'Wet lab 1: Benzonase'.
Whole Blood
- Centrifuge whole blood (with heparin) for 10 minutes at 2400 rpm (calculate appropriate g-force).
- Transfer 500 μL of plasma into an Eppendorf centrifugal filter (0.8 μm) and centrifuge for 5 minutes at 2000 x g.
How to aliquot the IC+HMB?
ALIQUOTING IC+HMB (host samples is NOT cat or dog)
! The internal control (IC) contains viral particles, so repeated freeze thawing should be avoided, the IC should be kept on ice as much as possible when thawed.
The 15 ml tube contains IC for about 300 aliquots of 46,5 μL.
- Aliquot the IC in 1,5 mL tubes. Label these tube in advance to speed up the process.
- Do the aliquoting in a laminar flow to prevent contamination of the IC.
- Thaw the 15 ml tube of IC on ice, once thawed aliquot the IC in 1,5 ml tubes (46,5 μl).
- I use a repetitive pipet (eg this type) to speed up the process.
- Aliquot the whole tube at once, do not put it back in the freezer once thawed.
- Keep the tube on ice as much as possible during aliquoting.
- Put the aliquots in the -70/-80°C freezer as soon as possible, I prepare some racks with open tubes, aliquot them, put the rest of the 15 ml tube back on ice, and put the aliquots in the freezer, repeat until the whole tube is aliquoted.
How to dilute the EDTA?
In the PathoSense reagents package is 1 µM EDTA (1 mL) included. This concentrated solution must be diluted to 10nM (100x dilution) in PCR graded water.
What to do with REF EXP-CTL001 included in the Minion Starter Pack?
REF EXP-CTL001 is a positive control included in the Minion Starter Pack. You do not need it for the validation nor the diagnostic run.
How to store the Zymo Extraction kit?
Store the Zymo Extraction kit at room temperature.
Find here more info.
Rapid Adapter Auxiliary V14 (ref. XP-RAA114) and Flow Cell Priming Kit (ref. XP-FLP004) are not included in Minion Mk1D Pack?
Rapid Adapter Auxiliary V14 (ref. XP-RAA114) and Flow Cell Priming Kit (ref. XP-FLP004) are not included in Minion Mk1D Pack. These reagents are not necessary to run the flow cell, but are additional reagents.
Normally, all reagents you need are in the Rapid Barcoding Kit 24V14 (SQK-RBK114.24).
Why I need the Flow Cell Wash Kit (ref.EXP-WSH004)?
The Wash Kit provides the reagents required to wash and reuse Flow cells.
This kit is recommended for users who:
- Want to wash flow cells and rerun with fresh samples.
- Wish to run samples on demand, rather than batching samples.
- Have accumulated a high number of "unavailable" pores during a previous run and wish to revert these pores to the "single pore" state.
FAQ Dry Lab
How long should we store our data?
It is important to store the pod5 and the fastq data at least one month after sequencing. This ensures re-sending or re-basecalling the data can still be repeated if necessary.
What to do when your run did not finish successfully? E.g. caused by insufficient storage, a technical error or manual interruption of the run.
Contact us through info@pathosense.com to let us know the data has not been transferred.
We can discuss together on how to proceed. This can result in a few different scenarios.
- If the sequencing was interrupted near the end of the sequencing, it might be worth still analysing the data. We will send you instructions on how to send the data of the failed run.
- If the sequencing was interrupted near the start of the sequencing, the issues should be resolved and the run sequencing run should be restarted for 9 hours.
What to do when the runfile was not added before the run has finished?
Contact us through info@pathosense.com to let us know the data has not been transferred. Follow these instructions to initiate the data transfer:
- Add the runfile in the correct folder cfr. the protocol (Part VI - RUN SET-UP)
- Move (cut-paste) the sequencing run folder from the monitored path (PathoSense_Diagnostics) to another location on the PC (e.g. the home folder).
- Move (cut-paste) it back to the PathoSense_Diagnostics path.
It is very important to cut-paste and not copy-paste the folder. When you copy the folder, you can not control the order in which the files arrive in the target folder. This means the report*.md file could arrive before the sequencing data. In that case, the CloudLink will be triggered to start the file transfer while the data is still incomplete.
What if the wrong output folder was chosen when setting up the run?
Check the available pores on your flowcell in MinKNOW (Experiments > Available pores). If more than 800 pores are still available, stop the run and restart the run in MinKNOW (again for 9 hours) with the correct output folder. Sufficient data will be generated in the following 9 hours, and the data transfer will then run without further manual steps.
If less than 800 pores are available, let the sequencing completely finish in the wrong output folder. Contact info@pathosense.com to let us know the data will be delayed. After the run has completely finished (since the run takes 9 hours, this will be the following day), cut-paste (NOT copy-paste) the sequencing folder to the PathoSense_Diagnostics folder. This will trigger the CloudLink to upload the data.
It is very important to cut-paste and not copy-paste the folder. When you copy the folder, you can not control the order in which the files arrive in the target folder. This means the report*.md file could arrive before the sequencing data. In that case, the CloudLink will be triggered to start the file transfer while the data is still incomplete.
Do not start moving the output files while sequencing! MinKNOW will just keep outputting files to the wrong output folder.
When should we do MinKNOW updates?
Do not update the MinKNOW software as long as everything is running smoothly. The newest MinKNOW updates can be quite unstable. So, if prompted to update, choose ‘skip this version’.
Who should we contact when we encounter MinKNOW issues?
Contact NanoPore’s customer support to solve issues related to flowcells or MinKNOW software, but add us in cc to keep us updated on the issues you are experiencing. You can store a loaded flowcell in the fridge for up to 1 week while you resolve the issues.
What to do when the wrong barcoding kit was selected for the sequencing run?
If you are not seeing any data getting generated in the barcodes you added, but all of your sequencing output shows up as ‘unclassified’, this could indicate that the wrong barcoding kit was selected when setting up the run.
In MinKNOW, check if the Kit ID is SQK-RBK114.24. If not, the wrong kit was selected during the set-up.
Check the available pores on your flowcell in MinKNOW (Experiments > Available pores). If more than 800 pores are still available, stop the run and restart the run in MinKNOW (again for 9 hours) with the correct kit selected. Sufficient data will be generated in the following 9 hours, and the data transfer will then run without further manual steps.
If less than 800 pores are available, let the sequencing completely finish. Contact info@pathosense.com to let us know the data will be delayed. After the run has completely finished (since the run takes 9 hours, this will be the following day).
Follow these instructions to repeat the basecalling with the correct kit and initiate data transfer.
- Delete everything in the fastq_pass folder from the sequencing directory.
- After the run has completely finished (since the run takes 9 hours, this will be the following day). Delete everything in the fastq_pass folder from the sequencing directory.
- Rebasecall the data
Start > Post sequencing analysis > Basecalling
Select the pod5 folder as the input folder > Continue
Select the folder that contains the pod5 folder and the sequencing reports as the output folder > Continue
Flow cell product: FLO-MIN114
Chemistry: DNA - 400bps - 5kHz
Model: Super-accurate basecalling
Modified basecalling: None
Minimum Q score: 1
> Continue
Barcoding kits: SQK-RBK114-24
Enable ‘Trim barcodes’
> Continue
> Continue
> Start
- The basecalling will output the files in a folder named “pass”. Once the basecalling has finished, rename the “pass” folder to “fastq_pass”.
- Move (cut-paste) the sequencing run folder from the monitored path (PathoSense_Diagnostics) to another location on the PC (e.g. the home folder).
Move (cut-paste) it back to the PathoSense_Diagnostics path.
What to do when the wrong barcodes were selected in the runfile / wrong runfile?
If you notice the wrong runfile was added while the sequencing is still running, just replace the file by the correct runfile. This will not cause any issues for further analysis as the runfile is only read in when the run has finished.
If you notice the wrong runfile was added after the sequencing has finished, contact info@pathosense.com to let us know the wrong barcodes were selected in the runfile. (Usually, we will see this in the output since some barcodes will be empty or the animal species does not match the output.)
- In the sequencing folder, replace the runfile with the correct one.
- Move (cut-paste) the sequencing run folder from the monitored path (PathoSense_Diagnostics) to another location on the PC (e.g. the home folder).
- Move (cut-paste) it back to the PathoSense_Diagnostics path.
This will trigger the CloudLink to upload the data again, so now the correct data will be sent to PathoSense.
It is very important to cut-paste and not copy-paste the folder. When you copy the folder, you can not control the order in which the files arrive in the target folder. This means the report*.md file could arrive before the sequencing data. In that case, the CloudLink will be triggered to start the file transfer while the data is still incomplete.
Will CloudLink keep running when I restart the computer?
Yes, CloudLink automatically restarts on reboot.
I want to do other sequencing runs on the same computer. How do I make sure the data is not sent to PathoSense?
CloudLink will only send data when it is:
- Located in the PathoSense_Diagnostics folder
- It contains a runfile in the correct location
So, simply setting the output folder to a different folder than the PathoSense_Diagnostics folder or not adding a runfile to the sequencing output will make sure data does not get transferred.
How many pores do you need to start the run?
Do not start sequencing with flowcells that fall below warranty (< 800 active pores). Nanopore will give you a replacement for these flowcells when you return them.
Can I reuse a flow cell?
xx
What is the minimum and maximum number of samples per run?
xx
Can we do sequencing for PathoSense on a Windows machine?
No, while MinKNOW can run on Windows, the data transfer through CloudLink is only supported on a Linux Machine.
Is a GPU NVIDIA RTX 4070 the minimum requirement?
FAQ PathoCloud
Where to find the 'Join Company' code?
How to register a sample in the lab?