Robotic Centrifugal Microfluidic Automation Enables Library Preparation of Nine Samples with a Tenfold Reduction in Reagent Consumption for Decentralized Sequencing

Abstract Next-generation sequencing (NGS) has become a crucial tool for medical diagnostics in decentralized laboratories. While sequencing and bioinformatics themselves employ standardized pipelines, wet laboratory workflows in these settings still rely on manual pipetting and sample handling. In particular, the library preparation procedures represent a critical bottleneck, accounting for up to 50% of total NGS costs and involving complex steps. Automation offers potential solutions to these challenges. However, existing automation approaches have limitations in satisfying throughput, investment costs, costs per sample, and degree of automation. Here, we implement a robotic centrifugal microfluidic platform around the Illumina Nextera XT DNA Library Preparation Kit or RoCM-LP platform, which supports library generation from microbial genomes. The RoCM-LP platform combines a centrifugal microfluidic-based cartridge with a pipetting robot, capable of producing nine libraries per run at low-medium initial investment costs ($50k–70k). The costs per sample were substantially reduced due to a tenfold decrease in reagent consumption in comparison to the manual reference workflow, and the number of manual handling steps was minimized from 45 to just three, namely reagent preparation (placing required reagents into the RoCM-LP platform), sample loading, and library retrieval. The approach achieved quality metrics comparable to those of the manual reference workflow and met all required sequencing quality thresholds across four Mycobacterium tuberculosis complex (MTBC) strains including drug-susceptible, BCG vaccine (Pasteur Bacillus Calmette–Guérin), multidrug-resistant (MDR), and extensively drug-resistant (XDR) strains for tuberculosis (TB) samples investigated, with an average coverage depth of 107, an average mapped read percentage of 97%, and an average coverage breadth percentage of 98%.