Loading multiple PromethION Flow Cells
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PromethION: Protocol
Loading multiple PromethION Flow Cells V PFC_9097_v1_revM_18Mar2020
For Research Use Only
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Priming and loading multiple flow cells
Washing multiple flow cells
Overview
For Research Use Only
1. Overview of the protocol
Flushing and washing flow cells at high scale
This protocol is a guideline to use alongside our automation protocols for PromethION to illustrate how to load, prime and wash up to 48 flow cells on a single device. Processing multiple flow cells can be time consuming and our protocols for sequencing kits are written for individual flow cells.
We estimate ~1 hour is saved using this protocol as it minimises pipette changes, including tip and volume alterations between steps and makes the most of the incubation time by completing each step per flow cell.
The user can adapt this protocol to any number of flow cells to fit their needs.
Priming and loading 48 flow cells takes approximately 1.5-2 hours. Washing, priming and re-loading 48 flow cells takes approximately 2-2.5 hours.
For information regarding the wash kit and the nuclease activity of the Flow Cell Wash Kit, please refer to the Flow Cell Wash Kit protocol.
2. Equipment and consumables
Materials
- DNA libraries
- Flow Cell Priming Kit V14 (EXP-FLP004)
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Flow Cell Wash Kit XL (EXP-WSH004-XL)
Consumables
- PromethION Flow Cell (FLO-PRO114M)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
Equipment
- PromethION sequencing device
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Ice bucket with ice
This protocol has been written for Kit 14 chemistry but can be used for all our library preparation protocols.
We recommend using this as a guideline for how to prepare multiple flow cells alongside the relevant protocol for your sequencing kit.
To wash your flow cells, the Flow Cell Wash Kit XL (EXP-WSH004-XL) is required.
To reload the flow cell with a DNA library, the following additional reagents are required.
- Flow Cell Tether (FCT)
- Flow Cell Flush (FCF)
- Library Beads (LIB) or Library Solution (LIS)
- Sequencing Buffer (SB)
- Elution Buffer (EB)
These can all be found in the Sequencing Auxiliary Vials V14 (EXP-AUX003). If only flow cell priming reagents are required, these can be found in the Flow Cell Priming Kit V14 (EXP-FLP004).
Flow Cell Wash Kit XL (EXP-WSH004-XL) contents
Name | Acronym | Fill volume per vial (µl) | Cap colour | No. of vials | No. of uses |
---|---|---|---|---|---|
Wash Mix | WMX | 150 | Brown | 1 | 48 |
Wash Diluent | DIL | 20,000 | White cap, brown stripe | 1 | 48 |
Storage Buffer | S | 25,000 | White cap, yellow stripe | 1 | 48 |
- Wash Mix (WMX) contains DNase I.
- Wash Diluent (DIL) contains the exonuclease buffer that maximises activity of the DNase I.
- The Storage Buffer allows flow cells to be stored for extended periods of time.
Sequencing Auxiliary Vials V14 (EXP-AUX003) contents
Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
---|---|---|---|---|
Elution Buffer | EB | Black | 2 | 500 |
Sequencing Buffer | SB | Red | 2 | 700 |
Library Solution | LIS | White cap, pink label | 2 | 600 |
Library Beads | LIB | Pink | 2 | 600 |
Flow Cell Flush | FCF | Light blue label | 2 | 8,000 |
Flow Cell Tether | FCT | Purple | 2 | 200 |
Flow Cell Priming Kit (EXP-FLP004) contents
Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
---|---|---|---|---|
Flow Cell Flush | FCF | 6 | Clear cap, light blue lable | 8,000 |
Flow Cell Tether | FCT | 1 | Purple | 200 |
3. Priming and loading multiple flow cells on a PromethION
Materials
- Elution Buffer (EB)
- Sequencing Buffer (SB)
- Library Beads (LIB)
- Library Solution (LIS)
- Flow Cell Flush (FCF)
- Flow Cell Tether (FCT)
Consumables
- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
Equipment
- PromethION sequencing device
- PromethION Flow Cell Light Shield
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
Loading multiple PromethION Flow Cells
To load libraries into multiple PromethION Flow Cells as efficiently as possible, we recommend completing each step of the protocol per flow cell, following the same order of flow cells for each step.
Note: PromethION Flow Cells are shipped with light shields already installed. We recommend keeping the light shields on the flow cell to save having to intall them after loading your library.
For example, if flow cells in positions 1A, 1B and 1C are loaded into the device sequentially, the next step is completed in the same order.
Below is an animation for how we recommend loading a full column of eight flow cells. This can be applied to any number of flow cells. In the animation, we illustrate how to complete the step for one flow cell and highlight the next flow cell the step is performed on, to give a quick overview of the entire process.
IMPORTANT
Scale up reagent volumes as needed
We recommend preparing enough reagents for the total number of flow cells being processed, including an extra two flow cells' worth as excess to take into account the extra volume required for pipetting errors.
Using the Library Solution
For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).
IMPORTANT
After taking flow cells out of the fridge, wait 20 minutes before inserting the flow cell into the PromethION for the flow cell to come to room temperature. Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
Insert all flow cells into the docking ports within the PromethION:
- Line up the first flow cell in position 1A with the connector horizontally and vertically before smoothly inserting into position.
- Press down firmly onto the flow cell and ensure the latch engages and clicks into place.
- Repeat for the next flow cell until all flow cells are loaded.
IMPORTANT
Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.
If not already completed, perform a flow cell check on all flow cells.
Please refer to the Flow Cell Check protocol for further information.
TIP
Reagents can be thawed and prepared during the flow cell check.
Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature, before mixing by vortexing. Then spin down before storing on ice.
Prepare the flow cell priming mix in a suitable vial for the number of flow cells to flush. Once combined, mix well by vortexing and keep at room temperature.
Reagents | Volume X1 flow cell | Volume X24 flow cells, including excess | Volume X48 flow cells, including excess |
---|---|---|---|
Flow Cell Tether (FCT) | 30 µl | 780 µl | 1,500 µl |
Flow Cell Flush (FCF) | 1,170 µl | 30,420 µl | 58,500 µl |
Total | 1,200 µl | 31,200 µl | 60,000 µl |
Slide the inlet port cover clockwise to open the inlet port of the first flow cell inserted. Repeat for all flow cells.
IMPORTANT
Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the inlet port, draw back a small volume to remove any air bubbles for all flow cells:
- Set a P1000 pipette tip to 200 µl.
- Insert the tip into the inlet port of the first flow cell.
- Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.
- Repeat for all the flow cells using the same pipette tip.
Complete the first flush as follows for all flow cells:
- Load 500 µl of the priming mix into the first flow cell via the inlet port, avoiding the introduction of air bubbles.
- Start a timer for 5 minutes. This is to ensure 5 minutes passes for the first flow cell before the second flush is started.
- Immediately repeat step 1 for the remaining flow cells using the same pipette tip.
Note: After each column (8 flow cells), thoroughly mix the priming mix to ensure it is homogenous.
Complete a second flush once all the flow cells have been flushed once, as follows:
- Check that the timer has finished to ensure it has been at least 5 minutes since the first flow cell was flushed.
- Load 500 µl of the priming mix into the inlet port of the first flow cell, avoiding the introduction of air bubbles.
- Repeat the previous step for the remaining flow cells in the same order as the first flush, using the same pipette tip.
Note: After each column (8 flow cells), thoroughly mix the priming mix to ensure it is homogenous.
Freshly prepare the DNA libraries for loading as outlined in the relevant protocol.
Note: Ensure you follow the relevant library preparation protocol to prepare your final library to ensure the compatible reagents and volumes are followed.
Load your samples:
- Gently mix the first library and load the full volume through the inlet port of the first flow cell.
- Using a new tip between each flow cell, repeat for the remaining flow cells, ensuring to gently mix each library before loading.
Note: For Kit 14, library volume is 200 µl. For previous kit chemistries, library volume is 150 µl.
Close the valve to seal the inlet port.
IMPORTANT
Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.
We recommend leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.
If the light shield has been removed from the flow cell, install the light shield as follows:
- Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
- Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.
END OF STEP
Close the PromethION lid when ready to start a sequencing run on MinKNOW.
Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.
For multiple flow cell washing, use the same experiment name and identifying sample IDs for all runs to enable all flow cells to be paused simultaneously.
4. Washing multiple flow cells on a PromethION
Materials
- Flow Cell Wash Kit XL (EXP-WSH004-XL)
Consumables
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
Equipment
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Ice bucket with ice
Preparation to run the washing procedure
An accumulation of pores in the 'recovering' state can occur ('unavailable' in the figure below). This reduces the rate of data acquisition due to fewer pores being available to accept and sequence strands. These pores can be reverted back to the 'active pore' state by pausing sequencing and washing the flow cell with the Flow Cell Wash Kit XL (EXP-WSH004-XL) (Figure 1). The wash step is only recommended when channels are lost to the 'recovering/unavailable' state. Further information is available in the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) protocol.
Figure 1. A flow cell has been loaded with a DNA library that has accumulated pores in the 'unavailable' state. The red asterisks indicate when sequencing has been paused and a wash step has been performed. After a wash step was completed, a significant number of pores reverted to the 'single pore' state and were available for sequencing.
- This protocol assumes that the flow cells have already had a DNA/RNA library run on them
- The aim is to remove most of the initial library and prepare the flow cells for the loading of a subsequent library
- The Wash Kit contains all solutions required for the removal of the initial library
- Data acquisition in MinKNOW should be stopped (if loading a new library or storing the flow cells), or paused (if loading more of the same library after the wash) during the wash procedure and also during subsequent library addition
- After the flow cells have been washed, a new library can be loaded, or the flow cells can be prepared for storage
TIP
We recommend keeping the light shield on the flow cell during washing if a second library will be loaded straight away.
If the flow cell is to be washed and stored, the light shield can be removed.
IMPORTANT
Scale up reagent volumes as needed
We recommend preparing enough reagents for the total number of flow cells being processed, including an extra two flow cells' worth as excess to take into account the extra volume required for pipetting errors.
IMPORTANT
A P1000 pipette must be used for all flushing steps to create a seal with the flow cell ports.
Place the tube(s) of Wash Mix (WMX) on ice. Do not vortex the tube.
Thaw the tube(s) of Wash Diluent (DIL) at room temperature.
Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.
Prepare the Wash Mix in a suitable vial for the number of flow cells to wash. Mix well by pipetting, and place on ice. Do not vortex the tube.
Reagent | Volume per flow cell | Volume X24 flow cells, including excess | Volume X48 flow cells, including excess |
---|---|---|---|
Wash Mix (WMX) | 2 μl | 52 μl | 100 μl |
Wash Diluent (DIL) | 398 μl | 10,348 μl | 19,900 μl |
Total | 400 μl | 10,400 μl | 20,000 μl |
Stop or pause the sequencing run in MinKNOW for all flow cells to be washed. Leave the flow cells in the device.
IMPORTANT
It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Remove the waste buffer, as follows:
- Ensure the inlet port is closed.
- Insert a P1000 pipette into a waste port and remove the waste buffer
Note: As the inlet port is closed, no fluid should leave the sensor array area.
Slide the inlet port cover clockwise to open the inlet port of the first flow cell inserted. Repeat for all flow cells.
IMPORTANT
Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the inlet port, draw back a small volume to remove any air bubbles:
Set a P1000 pipette to 200 µl.
For the first flow cell, insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer/liquid entering the pipette tip.
Repeat for the remaining flow cells using a __new pipette tip__.
Slowly load 200 µl of the prepared flow cell wash mix into the inlet port, as follows:
- Using a P1000 pipette, take 200 µl of the flow cell wash mix
- Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
- Set a timer for a 5 minute incubation.
Once the 5 minute incubation time is complete, carefully load the remaining 200 µl of the prepared flow cell wash mix into the inlet port, as follows:
- Using a P1000 pipette, take 200 µl of the flow cell wash mix
- Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
- Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Close the inlet port and start a 60 minute timer once the first flow cell has been loaded.
Repeat the two-step addition of the wash mix with the 5 minute break in between and the hour incubation for all of the flow cells.
After each row (8 flow cells), thoroughly mix the Wash Mix.
IMPORTANT
It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
For each flow cell, remove the waste buffer as follows:
- Ensure the inlet port is closed.
- Insert a P1000 pipette into a waste port and remove the waste buffer
Note: As both the inlet port is closed, no fluid should leave the sensor array area.
END OF STEP
Follow one of the two options described in the next steps of the protocol.
- Run a second library on the flow cell straight away
- Store the flow cell for later use
5. To run a second library on multiple PromethION Flow Cells straight away
Materials
- Flow Cell Wash Kit XL (EXP-WSH004-XL)
- Flow cell priming reagents available in your sequencing kit or in the following kits:
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Flow Cell Priming Kit V14 (EXP-FLP004)
Consumables
- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
Equipment
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Ice bucket with ice
IMPORTANT
Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.
We recommend leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.
TIP
The buffers used in this process are incompatible with conducting a Flow Cell Check prior to loading a subsequent library. However, the first pore scan once a sequencing run has started will report the number of nanopores available.
To run a second library straight away, follow the instructions in the 'Priming and loading multiple flow cells on a PromethION' of this protocol, with the recommendations below.
Note: For more consistent and better performance after a flushing a flow cell with the Flow Cell Wash Kit, we recommend the following:
- Pipette very slowly when loading priming mix into the flow cell.
- Wait five minutes between priming mix flushes.
- After the five minute pause, close the priming port, ensure the SpotON port is closed and remove the waste from waste port 1. This prevents the nuclease from diffusing through the flow cell.
The flow cells will already be inserted in the device and will not need a second flow cell check but will require priming with flow cell priming reagents. Once all the flow cells have been primed, either resume your run in MinKNOW or start a new sequencing experiment using Join existing experiment to use the same settings that were used previously.
Reloading a library
Additional buffers are required for reloading a library following the washing of a flow cell. These can be found in the one of the following expansion kits:
Sequencing Auxiliary Vials V14 (EXP-AUX003). This expansion contains vials of Sequencing Buffer (SB), Elution Buffer (EB), Library Solution (LIS), and Library Beads (LIB) with Kit 14 flow cell priming reagents: Flow Cell Flush (FCF) and Flow Cell Tether (FCT). This expansion is only compatible with our Kit 14 chemistry e.g. SQK-LSK114.
Flow Cell Priming Kit (EXP-FLP004). This expansion contains both Kit 14 flow cell priming reagents required: Flow Cell Flush (FCF) and Flow Cell Tether (FCT). This expansion is only compatible with Kit 14 chemistry.
For our previous chemistries:
Sequencing Auxiliary Vials expansion (EXP-AUX001). This expansion contains vials of Elution Buffer (EB), Sequencing Buffer (SQB), and Loading Beads (LB), additional to those found in DNA sequencing kits for our Kit 9 chemistry.
Sequencing Auxiliary Vials expansion (EXP-AUX002). This expansion contains vials of Sequencing Buffer II (SBII), Elution Buffer (EB), Loading Solution (LS), and Loading Beads II (LBII), additional to those found in Kit 10 and 11 chemistry, such as:
- Kit 10 e.g. SQK-LSK110
- Kit 11 e.g. SQK-PCS111
- Flow Cell Priming Kit (EXP-FLO002). This expansion contains both flow cell priming reagents required: Flush Buffer (FB) and Flush Tether (FLT). This expansion is compatible with Kit 9, 10 and 11 chemistry.
IMPORTANT
When priming a flow cell after a nuclease wash with the Flow Cell Wash Kit, it is vital to wait five minutes between the priming mix flushes and to remove the waste for effective removal of the nuclease.
TIP
Library storage recommendations
We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, reloading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes. For further information, please refer to the DNA library stability Know-How document.
6. To store multiple PromethION Flow Cells for later use
Materials
- Storage Buffer (S)
Consumables
- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
Equipment
- P1000 pipette and tips
Storage Buffer (S) can be used to flush flow cells for storage for later use or to check number of available nanopores before loading another library.
IMPORTANT
Scale up reagent volumes as needed
We recommend preparing enough reagents for the total number of flow cells being processed, including an extra two flow cells' worth as excess to take into account the extra volume required for pipetting errors.
Thaw Storage Buffer (S) at room temperature.
Mix contents thoroughly by pipetting and spin down briefly.
Slide the inlet port cover clockwise to open the inlet port of the first flow cell inserted. Repeat for all flow cells.
IMPORTANT
Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the inlet port, check for a small air bubble under the cover. Draw back a small volume to remove any air bubbles:
Set a P1000 pipette to 200 µl.
For the first flow cell, insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.
Repeat for the remaining flow cells.
Slowly add 500 μl of Storage Buffer through the inlet port of the first flow cell and repeat for the remaining flow cells.
After each row (8 flow cells), thoroughly mix the Storage Buffer by pipetting.
Once all the flow cells are loaded, close the inlet port covers, and remove any buffer from the waste port.
The waste liquid can be aspirated from either one of the ports, labelled 2 and 3 on the flow cell.
Remove the flow cells from the device by pressing down on the latch to release the flow cells before sliding out smoothly.
Store the flow cells at 4-8° C.
END OF STEP
When you wish to reuse the flow cell, remove the flow cell from storage, and allow it to warm to room temperature for ~5 minutes.
IMPORTANT
After performing a flow cell wash or storing your flow cell, we recommend using running a 'Flow cell check' to check number of available nanopores.
Load your flow cell into the device with Storage Buffer (S) and start a Flow cell check to detect the number of active pores. For more information, please visit the Flow cell check section of our MinKNOW protocol.
After the Flow cell check, prime your flow cell and load the library before starting a new sequencing run.