DIYNAFLUOR: An Affordable Plug-and-Play DIY Nucleic Acid Fluorometer for Quantification of eDNA Sequencing Libraries in Resource Limited Settings

Nucleic Acid (NA) fluorometry is widely employed for quantifying environmental DNA (eDNA) samples and their downstream DNA sequencing libraries, owing to its sensitivity, accuracy, and speed. However, with commercial models priced between $2.5k-4.5k USD, NA fluorometers present a significant cost barrier to eDNA sequencing in resource-limited settings (RLSs), especially when contrasted against Oxford Nanopore Technologies' $3k USD MinION portable Nanopore sequencing platform. To address this barrier, we have developed the "DIYNAFLUOR" (DIY Nucleic Acid FLUORometer), a portable, open-source, <$40 USD NA fluorometer, designed using off-the-shelf components, simple 3D-printed parts, and plug-and-play, solder-free assembly. Compatible with the popular DNA-centric Qubit Assay kits, the DIYNAFLUOR notably demonstrated an 'in-assay' Limit of Detection with the Qubit High Sensitivity kit of 0.0028 ng/uL, and, using a 2-point linear calibration methodology, an average absolute bias of 0.018 ng/uL across a 0-10 ng/uL working regime. Verification was performed through comparative measurements against a Qubit 4 fluorometer in a busy biotechnology laboratory, and build instructions were validated through assembly and qualification of three DIYNAFLUORs by researchers outside the design team. To demonstrate its practical application for RLS eDNA analysis, the DIYNAFLUOR was used to perform all QC measurements throughout the preparation of 16S and 18S metabarcoding libraries generated from eDNA extracted from Australian lake water, leading to the successful identification of Australian fauna via Nanopore sequencing. Finally, configurations of the DIYNAFLUOR for RNA and Protein quantification and a <13 cents USD per-measurement DNA assay based on SYBR Safe dye are briefly described.