B-025 Revolutionizing NGS-driven Laboratory Developed Tests: The impact of automated liquid handling on plate-based bead clean-up
Authors: Henry L , Pearson A , Ota A 
Published: October 2024

Abstract

Background
Automated liquid handling systems are proving to be a transformative technology in the landscape of Laboratory Developed Tests (LDTs), especially those utilizing Next-Generation Sequencing (NGS). By enhancing accuracy, boosting efficiency, and offering scalable solutions, these systems enable labs to meet current and future challenges more effectively. The right system not only improves the quality of diagnostics but also positions labs to be agile and adaptive in a rapidly advancing healthcare environment. firefly® is a well-suited liquid handling solution for diagnostics labs developing LDTs due to its compact footprint, user-friendly software and exceptional versatility. Validated protocol templates that support NGS library preparation are readily available from the firefly cloud and can be adapted to meet specific lab needs. For example, automated bead-based clean-ups designed for 96- and 384- NGS applications are available on firefly. These remove DNA fragments of undesirable sizes, enzymes or primers rom the amplified DNA after a PCR reaction. Automating this process facilitates higher throughput and scaling. firefly has a unique combination of 6 positive displacement dispense heads and a 96/384-well air-displacement pipetting head, making it ideal to distribute the reagents required for bead clean-ups.

Methods
firefly protocols were developed and optimized to be compatible with a range of elution volumes and bead ratios. Protocol performance was tested using library DNA (∼300bp). The same input DNA was plated out and quantified using the QuantiFluor dsDNA system (Promega) before and after running the protocol. This data was used to assess any reduction in DNA concentration and uniformity across the plate. The protocol's ability to successfully modulate the fragment size distribution was demonstrated using a 50bp DNA ladder and different bead ratios. Data was obtained using a Fragment Analyzer (Agilent, DNF-474 HS NGS Fragment Kit). Well-to-well contamination was assessed by running no-template-controls (NTCs) in wells adjacent to library DNA, then analyzed by qPCR using a LightCycler 480 System (Roche, KAPA Library Quantification kit).

Results
96 format has a setup time of 10 minutes, run time of 43 and %CV library concentration of &lt;6%.
384 format has a setup time of 10 minutes, run time of 47 and %CV library concentration of &lt;8%.
Uniformity of concentration and fragment sizes was seen across the plates. Comparison of the library concentration before and after the clean-up showed a 5% reduction in DNA concentration after the 96-well plate clean-up and an 11% reduction in library concentration when using the 384-well plate clean-up. Fragment size analysis showed that fragment sizes can be successfully modulated on firefly by changing the bead ratio of the firefly run and no detectable well-to-well contamination was detected by qPCR.

Conclusions
We have demonstrated that automated bead clean-ups on firefly are uniform show uniformity across both 96- and 384-well plates, with no detectable well-to-well contamination, to deliver the consistent and robust results required for an LDT. Bead-based cleanups on firefly offer a fast, 100% walkaway solution. By running 96-samples at once on firefly, we see approximately a 6-fold increase in throughput than when performed manually by a single user.