The VolTRAX V2b is a portable, multi-purpose device designed to automatically prepare DNA or RNA libraries ready for analysis on a nanopore sequencing platform. The device is compact and USB-powered, to allow portability of library preparation and sequencing in a laboratory or non-laboratory environment.

The VolTRAX V2b with a disposable cartridge inserted.

The VolTRAX V2b device along with the cartridge allows a user to choose a library preparation protocol, load their sample with supplied reagents and press go. The device prepares the library following a series of steps programmed by the software.

The device includes heating elements for incubations and PCR, and magnetic elements for bead-based separations. These features combine with the VolTRAX capabilities to precisely move, mix and incubate droplets to enable both basic and complex sample preparation methods to be carried out with a click of a button.

VolTRAX V2b is simple to use and needs only minutes of hands-on time to prepare a library.

The VolTRAX Sequencing Kit (VSK-VSK004) and VolTRAX Multiplex Kit (VSK-VMK004) protocols for VolTRAX V2b require clean, extracted DNA that is of high molecular weight, accurately-quantified and free from contaminants and detergents. The DNA should be prepared in 10 mM Tris-HCl, pH 8.0.

The VolTRAX PCR Tiling 1-12 COVID-19 Kit (VSK-PTC001) and VolTRAX RT-PCR Sequencing Kit 1-12 protocol requires total RNA in 10 mM Tris-HCl, pH 8.0, extracted from samples that have been screened by a suitable qPCR assay. Samples must have a Ct value of 30 or less, and be free from contaminants and detergents.

Contaminants

Contaminants can impede the function of the library preparation chemistry for VolTRAX V2b. For information about the impact of contaminants on reaction efficiency, see the Contaminants Know-How piece.

VolTRAX V2b is driven by the VolTRAX software, which is responsible for the control of the device and consumable cartridge array.

VolTRAX V2b computer requirement is less demanding than a MinION™. If you are currently running a MinION Mk1B on Windows, or you plan to use a MinION Mk1B with VolTRAX, the computer requirements for your MinION Mk1B should be sufficient to operate the VolTRAX.

If you plan to purchase a system dedicated to VolTRAX V2b, the minimum requirements are:

† Only the USB TYPE C – Gen 3.1 (I or II) or higher – 5 V, 3 A can provide the full range of functionalities on VolTRAX V2b.

To ensure users can easily determine their VolTRAX status; a series of LEDs are included and described below.

Under-side LEDs:

The LEDs shown in the figure are indicators of the power supplied to the device.

1. Red - This indicates that the USB Type-C cable is correctly inserted into the port on the device. This is for Development use only.
2. Red - This LED signals that 900 mA are being supplied to the device; this is the least powerful type of USB-Type C or 3.1 Type I/II port. The device will operate basic protocols, but will not be able to perform thermal cycling required for PCR-based library preparations.
3. Blue - This LED signals that 1.5 A are being supplied to the device; this is a commonly occurring power supply of USB-Type C ports. The device will operate basic protocols, but will not be able to perform thermal cycling required for PCR-based library preparations.
4. Green - This LED signals 3 A are being supplied to the device; this is the requirement for the full capabilities of the VolTRAX V2B device.
5. Blue - This LED signals power to the device.
6. Blue - This LED signals power to the device.
7. These are for use by Oxford Nanopore Technologies.

It is particularly important that LED 4 (green) is illuminated, if the user requires the thermal cycler to prepare their library. If the LED is not on, 3 A are not being supplied to the device.

To check that the laptop to be used with the VolTRAX has a port that supplies 3 A and that the power rating is set to 3 A, contact your IT Department.

Internally at Oxford Nanopore Technologies, we have used USB-power meters as a way of measuring the current provided by a USB port when a laptop is running off charge or plugged in. Users may wish to look into these in advance of their VolTRAX devices arriving.

The Oxford Nanopore Technologies software will require access to the AWS IP ranges currently listed here:

http://docs.aws.amazon.com/general/latest/gr/aws-ip-ranges.html

Access to the following IP addresses is also needed:

              178.79.175.200  
               96.126.99.215  

VolTRAX software is under rapid development and we ask users to keep up-to-date with the latest software revisions.

Latest software versions will be available on our software downloads page.

Microsoft Windows updates

It is essential to check the settings for Windows updates. Windows Updates Automatic restarts need to be disabled and it is recommended that Windows Update installs are set to occur on shutdown only.

Below is an overview of the configuration experiment using a VolTRAX V2b with the Configuration Kit (VCK-V2003b).

1. Loading a protocol

• Use the protocol selector in the UI to load the VCK-V2003b protocol.
• When prompted, connect your VolTRAX V2b device, press Continue and await connection.

2. System QC - Cartridge Test

• When prompted, connect a cartridge to the VolTRAX device and press Continue to proceed to the Cartridge Test.

3. Running and loading VCS

• Follow instructions and advice on the equipment required and pipetting technique
• Prime the cartridge as instructed by the UI
• Load the VolTRAX Configuration Solution (VCS) and the VX AMPure XP Config (VXC) into the ports as instructed by the UI
• The solutions will be manipulated to indicate what the user should expect during a run

4. Extracting the VolTRAX Configuration Solution from the cartridge

• Follow the extraction instructions, using UI as a visual guide, to slowly extract the VCS
• Discard the extracted VCS

Once an option is selected from the menu, the UI will show a preview of the steps involved in library preparation. The example below is for the Configuration Kit.

The VolTRAX V2b provides an automated process for preparing a DNA library. The chemistry of the library preparation is the same as for manual library preparation for a MinION or GridION sequencing experiment.

Below is an overview of the library preparation stages using a VolTRAX V2b, blue cartridge and the VolTRAX Sequencing Kit (VSK-VSK004).

1. Loading a protocol

• Use the protocol selector in the UI to load the VSK-VSK004 protocol.
• When prompted, connect your VolTRAX V2b device, press Continue and await connection.

2. System QC - Cartridge Test

• When prompted, connect a cartridge to the VolTRAX device and press Continue to proceed to the Cartridge Test.

3. Loading the Priming Fluid and reagents into the cartridge

• Prime the cartridge by twisting the oil container and removing the foil tab.
• The UI will recommend the quality, concentration, buffer and Absorbance (A) readings for your input DNA. These are stated at the stage of 'Reagent Loading' later in this protocol.
• Follow the instructions on the equipment required and pipetting technique.
• Load the appropriate reagents and extracted DNA into the ports as instructed by the UI.
• The reagents will be manipulated by the VolTRAX V2b to prepare the library for nanopore sequencing.

4. Extracting the library from the cartridge

• Follow the extraction instructions, using UI as a visual guide, to slowly extract the library from the cartridge, then transfer it into an Eppendorf tube.
• Prepare the library for loading onto the flow cell.

The VSK-VSK004 library preparation protocol applies our rapid chemistry, with a bead-based purification step.

Once an option is selected from the menu, the UI will show a preview of the steps involved in library preparation. The example below is for the Configuration Kit.

The VolTRAX V2b provides an automated process for preparing a DNA library. The chemistry of the library preparation is the same as for manual library preparation for a MinION or GridION sequencing experiment.

Below is an overview of the library preparation stages using a VolTRAX V2b and the VolTRAX Multiplex Kit (VSK-VMK004).

The VSK-VMK004 library preparation protocol uses our rapid chemistry, with a bead-based clean-up step. The kit contains ten barcodes for multiplexing up to ten samples in one library preparation.

1. Loading a protocol

• Use the protocol selector in the UI to load the VSK-VMK004 protocol.
• When prompted, connect your VolTRAX V2b device, press Continue and await connection.

2. System QC - Cartridge Test

• When prompted, connect a cartridge to the VolTRAX device and press Continue to proceed to the Cartridge Test.

3. Loading the Priming Fluid and reagents into the cartridge

• Prime the cartridge by twisting the oil container and removing the foil tab.
• The UI will recommend the quality, concentration, buffer and Absorbance (A) readings for your input DNA. These are stated at the stage of 'Reagent Loading' later in this protocol.
• Follow the instructions on the equipment required and pipetting technique.
• Load the appropriate reagents and extracted DNA into the ports as instructed by the UI.
• The reagents will be manipulated by the VolTRAX V2b to prepare the library for nanopore sequencing.

4. Extracting the library from the cartridge

• Follow the extraction instructions, using UI as a visual guide, to slowly extract the library from the cartridge, then transfer it into an Eppendorf tube.
• Prepare the library for loading onto the flow cell.

Once an option is selected from the menu, the UI will show a preview of the steps involved in library preparation. The example below is for the Configuration Kit.

Priming and loading: The steps for priming and loading the SpotON Flow Cell. Written instructions are given below. The library is loaded dropwise without putting the pipette tip firmly into the port.

Take care to avoid introducing any air during pipetting.

The VolTRAX PCR Tiling 1-12 COVID-19 Kit generates tiled PCR amplicons from the SARS-CoV-2 viral RNA for whole genome sequencing using the VolTRAX V2b platform to improve consistency in library preparation due to the precise fluid handling of the device.

This kit provides all the reagents required to generate reverse-transcribed and PCR-amplified material from SARS-CoV-2 RNA samples using VolTRAX automation to prepare the library, without the need to purchase further components. The PCR tiling generated overlapping amplicons across the whole SARS-CoV-2 genome before barcoding and pooling multiple samples together for sequencing.

Below is an overview of the library preparation stages using a VolTRAX V2b, blue cartridges and the VolTRAX PCR Tiling 1-12 COVID-19 Kit (VSK-PTC001).

1. Loading a protocol

• Use the protocol selector in the UI to load the VSK-PTC001 protocol.
• When prompted, connect your VolTRAX V2b device, press Continue and await connection.

2. System QC - Cartridge Test

• When prompted, connect a cartridge to the VolTRAX device and press Continue to proceed to the Cartridge Test.

3. Loading the Priming Fluid and reagents into the cartridge

• Follow the instructions on the equipment required and pipetting technique.
• Prime the cartridge by twisting the oil container and removing the foil tab.
• Load the appropriate reagents and extracted RNA into the ports as instructed by the UI.
• The reagents will be manipulated by the VolTRAX V2b to prepare the library for nanopore sequencing.

4. Extracting the library from the cartridge

• Follow the extraction instructions, using UI as a visual guide, to slowly extract the library from the cartridge, then transfer it into an Eppendorf tube.
• Prepare the library for loading onto the flow cell.

The VolTRAX PCR Tiling 1-12 COVID-19 Kit generates overlapping amplicons in a tiled fashion across the SARS-CoV-2 genome. First, the RNA samples are reverse-transcribed and then amplified by tiled PCR using separate primer pools. The primer pools are combined and SPRI purified before moving into the rapid barcoding stage which attaches Rapid Barcodes to the DNA ends for samples to be pooled.

The Board test should be carried out on your VolTRAX device before proceeding with PCR experiments. The most recent models of the VolTRAX V2b feature improved temperature calibration for PCR, and the Board test checks whether you have a version of the device that is PCR-compatible.

If the board test has been successful, you can proceed with PCR experiments.

If the board test has failed, please contact Support.

Once an option is selected from the menu, the UI will show a preview of the steps involved in library preparation. The example below is for the Configuration Kit.

The VolTRAX RT-PCR Sequencing Kit 1-12 generates PCR amplicons from the user's input RNA using the VolTRAX V2b platform to improve consistency in library preparation due to the precise fluid handling of the device.

This kit provides the reagents required to generate reverse-transcribed and PCR-amplified material from RNA samples using VolTRAX automation to prepare the library. The user provides their own PCR primers to generate custom amplicons, before barcoding and pooling multiple samples together for sequencing. We recommend using 10 mM Tris-HCl pH 8.0 as a buffer for the primers, and using the VolTRAX device to optimise the PCR conditions. When loading PCR primers onto the VolTRAX, we recommend loading at 3X the desired reaction concentration.

Below is an overview of the library preparation stages using a VolTRAX V2b, blue cartridges and the VolTRAX RT-PCR Sequencing Kit 1-12 (VSK-VPS001).

1. Loading a protocol

• Use the protocol selector in the UI to load the VolTRAX RT-PCR Sequencing Kit 1-12 protocol.
• When prompted, connect your VolTRAX V2b device, press Continue and await connection.

2. System QC - Cartridge Test

• When prompted, connect a cartridge to the VolTRAX device and press Continue to proceed to the Cartridge Test.

3. Loading the Priming Fluid and reagents into the cartridge

• Follow the instructions on the equipment required and pipetting technique.
• Prime the cartridge by twisting the oil container and removing the foil tab.
• Load the appropriate reagents, custom primers and extracted RNA into the ports as instructed by the UI.
• You will be given the option to adjust the PCR timings and temperatures to allow you to use custom primers.
• The reagents will be manipulated by the VolTRAX V2b to prepare the library for nanopore sequencing.

4. Extracting the library from the cartridge

• Follow the extraction instructions, using UI as a visual guide, to slowly extract the library from the cartridge, then transfer it into an Eppendorf tube.
• Prepare the library for loading onto the flow cell.

The VolTRAX RT-PCR Sequencing Kit 1-12 generates amplicons across target regions or organisms of interest within the extracted RNA sample. First, the RNA samples are reverse-transcribed and then amplified by PCR using the user-defined primers. The samples are purified with AMPure XP beads before tagmenting the DNA amplicons with the Rapid Barcodes, adding the sequencing adapter and pooling for sequencing.

The Board test should be carried out on your VolTRAX device before proceeding with PCR experiments. The most recent models of the VolTRAX V2b feature improved temperature calibration for PCR, and the Board test checks whether you have a version of the device that is PCR-compatible.

If the board test has been successful, you can proceed with PCR experiments.

If the board test has failed, please contact Support.

Once an option is selected from the menu, the UI will show a preview of the steps involved in library preparation. The example below is for the Configuration Kit.