B-016 Automated Next Generation Sequencing Fluidic-Based Device for Sample to Library Preparation: A Flexible Solution for Rapid Decentralized Sequencing for Infectious Disease Diagnostics

Abstract

Abstract Background In addition to the established use of Next Generation Sequencing (NGS), such as drug resistance monitoring in HIV, the recent COVID19 pandemic has shown the crucial need of NGS technologies as tools for identification of clinically relevant variants of the SARS-CoV-2 virus. Typically, in these settings, to be cost-effective most NGS analyses were conducted using larger batch sizes often resulting in long turn-around times of 2–3 weeks the need of trained personnel, and centralized testing facilities. Methods We have developed an easy to use automated fluidic-based benchtop device to bridge a perceived gap in NGS library preparation and provide a solution for decentralized testing that does not require specially trained personnel. With a batch size of 6, smaller hospitals and laboratories can perform NGS library preparation on fresh or archived samples with a fast turn-around-time of two days, facilitating earlier reporting to speed up diagnosis and treatment. The Helaxy NGS fluidic device performs sample nucleic acid extraction and a 2-step PCR in a plastic fluidic card, which is the sole consumable required for the extraction and library prep process.The sample is applied to the fluidic card by manual pipetting followed by loading of the card into the device. Nucleic acids are extracted with a combination of magnetic bead pull-down and centrifugal force in a purification chamber to give an eluate that is spun to a separate PCR chamber that uses the concept of thermal induction and cycling. Barcoding for library preparation follows within the same PCR chamber and the resulting product is transferred into another fluidic card for size selection purification, normalization and pooling to generate a library for input into compatible NGS sequencers, e.g., but not confined to, Illumina instruments. Results In order to demonstrate clinical utility we have shown that the Helaxy NGS fluidic device can successfully extract nucleic acid from at least three sample types: SARS-CoV-2 RNA from nasopharyngeal swabs, HIV RNA from plasma and 16S RNA from stools. Using in-house and commercial assays, we have generated libraries of target amplicons using the fluidic device workflow followed by sequencing with Illumina sequencers iSeq and MiSeq. Sequencing results analysed and reported by the Helaxy NGS Bioinformatics Reporting Pipeline revealed expected variants and mutations in the samples. We correctly identified all the unique mutations such as L211del and G446S in commercially available inactivated Omicron BA1 variant of the SARS-CoV-2 virus as well as characterised drug-resistant mutations such as L90M in the Protease gene and D67N and Y181C in the Reverse Transcriptase gene in commercially available inactivated HIV-1 virus. Conclusion The Helaxy NGS fluidic device can thus effectively provide an easy-to-use automated solution for sample library preparation in decentralized facilities to reduce the turn-around-time from sample collection to result reporting for clinical diagnosis and treatment.