Flow virometry for water-quality assessment: Protocol optimization for a model virus and automation of data analysis

Flow virometry (FVM) can support advanced water treatment and reuse by delivering near real-time information about viral water quality. But maximizing the potential of FVM in water treatment and reuse applications requires protocols to facilitate data validation and interlaboratory comparison—as well as approaches to protocol design to extend the suite of viruses that FVM can feasibly and efficiently monitor. In the npj Clean Water article “Flow virometry for water-quality assessment: Protocol optimization for a model virus and automation of data analysis,” we address these needs by first optimizing a sample-preparation protocol for a model virus (T4 bacteriophage) using a fractional factorial experimental design. We then compare manual and algorithmic methods of analyzing complex FCM data collected by applying the optimized protocol to (i) a clean solution spiked with a variety of biological and non-biological viral surrogates [mixed-target experiment], and (ii) tertiary treated wastewater effluent spiked with T4 bacteriophage and two sizes of fluorescent polystyrene beads [environmental spike experiment]. This repository contains the FCM data used to develop the optimized protocol and to test the two analytical methods.

Methods
All data were collected by analyzing a 10-mL volume of the sample in question using the 488 nm (blue) solid-state laser, the lowest possible instrument flowrate (5 mL/min), and a FITC = 800 threshold on a NovoCyte 2070V Flow Cytometer coupled with a NovoSampler Pro autosampler (Agilent). Green fluorescence (FITC) intensity was collected at 530 ± 30 nm; forward and side scatter (FSC and SSC) intensities were collected as well. For the optimization experiments, 10 mL of an unstained control was run after each sample. The instrument was flushed in between each sample and control by running 150 mL of 1x NovoClean solution (Agilent) followed by 150 mL of MQ water through the SIP at the highest instrument flow rate (120 mL/min). Instrument performance was ensured by performing the instrument’s built-in quality control (QC) test at least monthly. The FCM data were exported directly to .fcs (the standard format for flow cytometry/virometry data) files. All of the raw .fcs files used for the optimization experiments, mixed-target experiments, and environmental spike experiments are provided in this repository. For the mixed-target and environmental spike experiments, these .fcs files were then manually gated and exported to .csv files for use in downstream, algorithmically assisted analysis. Each of these .csv files is provided in this repository as well.