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

Poster

Date: 3rd December 2020

VolTRAX is capable of multiplexed amplification, quantification and preparation of sequencing libraries from biological samples, enabling consistent library quality even in non-laboratory environments

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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) VMK 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 using the VolTRAX Multiplexing Kit. We obtain very similar read counts from each barcode, showing the reproducibility of library preparation on the device (Fig. 2c). We have also incorporated fluorescence measurement into VolTRAX, allowing quantification (Fig. 2d).

Fig. 3 PCR on VolTRAX a) whole-genome PCR b) read length c) and d) CO1 species identification

Whole-genome PCR and library prep performed on VolTRAX

VolTRAX V2 has a Peltier block with a sufficiently fast temperature ramp rate to perform PCR amplification. We have developed a VolTRAX whole-genome PCR kit which, like the RBP kit uses a transposase to simultaneously fragment genomic DNA and to attach universal PCR adapters. VolTRAX amplifies complex mixtures of genomes with similar efficiency to when the reactions are performed in a tube (Fig. 3a) and with the expected read-length distribution (Fig. 3b). It is also possible to perform locus-specific PCR on the device. 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).

Fig. 4 Multiplexed tiled PCR, pool quantification and barcoded library preparation on VolTRAX

Multiplexed whole-genome SARS-CoV-2 PCR including on-device quantification

We have developed a VolTRAX protocol for whole genome sequencing of SARS-CoV-2 using a modified version of the ARTIC protocol (Fig. 4a). Viral RNA is loaded on to the device, reverse transcribed using random hexamer primers and PCR amplified to generate overlapping 400 base pair regions of the genome. These amplicons can be combined, quantified and prepared for sequencing using a transposase-based approach, all on the device. The protocol is designed to multiplex up to 4 different samples. VolTRAX V2 is able to quantify DNA. Here we show the fluorescence output of three positive samples and a negative control (Fig. 4b). The sequence reads show largely even coverage across the input sample genome. The dropout observed at regular 5 kb intervals is expected due to the RNA samples used (Fig. 4c).

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