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VolTRAX: a versatile, programmable, portable device for sample and library preparation

Poster

Date: 24th May 2018

VolTRAX is designed to extract nucleic acids and prepare sequencing libraries from biological samples, enabling consistent library preparation even in non-laboratory environments

Fig. 1 The VolTRAX device

VolTRAX: automated sample and library preparation without human intervention

Oxford Nanopore has developed VolTRAX – a small device designed to perform all of the molecular biological manipulations required to convert a raw biological sample to a form ready for analysis on a nanopore sensing device, without the need for human intervention (Fig. 1). Ultimately, the device will be dockable with MinION, GridION or PromethION, allowing fully automated sequencing in both laboratory and non-laboratory environments. The technology behind VolTRAX utilises an array of pixels. By applying a charge, reagent and sample droplets are moved in a path programmed by software, allowing the separate sample and library preparation processes to be performed sequentially.

Fig. 2 Library prep a) read-length distribution b) throughput c) multiplexing d) quantification

Library prep workflows show equivalent performance on VolTRAX and in tubes

We are adapting all of our library preparation protocols to work on VolTRAX. When a library is prepared on VolTRAX, we obtain a similar read-length distribution to that seen when the prep is performed in a tube (Fig. 2a). Additionally, the reactions have been optimised extensively on VolTRAX to give equivalent performance to the highest-throughput tube prep (Fig. 2b). Having several reagent ports on the VolTRAX cartridge allows us to perform multiple barcoded library preps in parallel. We obtain very similar read counts from each barcode, showing the reproducibility of library preparation on the device (Fig. 2c). We are also incorporating fluorescence measurement into VolTRAX, to allow quantification of DNA and RNA (Fig. 2d).

Fig. 3 Sample to result: using VolTRAX in species-identification workflows

Cell lysis, 16S PCR and library prep performed on VolTRAX, and CO1 species ID

We performed lysis of an E. coli culture, DNA purification using SPRI beads, 18 rounds of PCR on the extract to amplify the ~1.5 kb 16S region, and attachment of sequencing adapters (Fig. 3a). All steps were performed on VolTRAX. Finally, we removed the prepared library and sequenced it to confirm the identity of the species using our Epi2Me 16S species identification workflow (Fig. 3b). Some samples are more difficult to extract DNA from, and here it can be helpful to use bead-beating prior to DNA isolation. We extracted DNA from the invasive ladybird species Harmonia axyridis by bead-beating, and loaded the crude extract onto VolTRAX, where we amplified a 650 bp region of the CO1 gene and attached sequencing adapters (Fig. 3c). BLAST analysis of the reads confirmed the identity of the sample (Fig. 3d).

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Fig. 4 Whole-genome amplification on VolTRAX a) workflow b) WIMP identification

Whole-genome amplification on VolTRAX enables low-input sequencing

In situations where only small amounts of input DNA are available, whole-genome amplification (WGA) can be used to increase the mass of DNA. With this in mind, we adapted a WGA protocol for use on VolTRAX. In contrast to the standard protocol, a proteinase K digestion and SPRI clean-up are performed after the WGA reaction. The DNA is then fragmented and adapters are added using our rapid library prep protocol (Fig. 4a). We tested the protocol on a sample of soil taken from the Oxford Science Park, where the ONT labs are located. The soil was resuspended in water, filtered and the filtrate loaded onto VolTRAX for WGA, quantification and library preparation. As the library was being sequenced, we used the WIMP analysis tool to identify the species present, in real time. These included bacteria, fungi and viruses (Fig. 4b).

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