The history and conceptual framework of assays and screens

Abstract

Since the 16th century, assays and screens have been essential for scientific investigation. However, most methods could be significantly improved, especially in accuracy, scalability, and often lack adequate comparisons to negative controls. There is a lack of consistency in distinguishing assays, in which accuracy is the main goal, from screens, in which scalability is prioritized over accuracy. We dissected and modernized the original definitions of assays and screens based upon recent developments and the conceptual framework of the original definitions. All methods have three components: design/measurement, performance, and interpretation. We propose a model of method development in which reproducible observations become new methods, initially assessed by sensitivity. Further development can proceed along a path to either screens or assays. The screen path focuses on scalability first, but can later prioritize analysis of negatives. Alternatively, the assay path first compares results to negative controls, assessing specificity and accuracy, later adding scalability. Both pathways converge on a high-accuracy and throughput (HAT) assay, like next generation sequencing, which we suggest should be the ultimate goal of all testing methods. Our model will help scientists better select among available methods, as well as improve existing methods, expanding their impact on science.

Most synonymous allelic variants in HIV tat are not silent

“…There are three important considerations for this application of the dataset: 1, the GigaAssay is not a screen, rather it is an assay where both positives and negatives are directly measured with high accuracy and negatives are not inferred [ 33 ]; and 2, since the synonymous variants arose due to synthesis errors, there were fewer barcodes with UMIs than the >100 UMIs for each allelic variant previously reported, typically <6 for each mutant. Therefore, we only analyzed mutants that had at least 2 barcodes in at least one of the replicates and activities were averaged for technical replicates ( File S1 ).…”

Synonymous Variants of Uncertain Silence

“…In our prior research, we introduced a novel system known as the GigaAssay [ 18 , 19 ], which we have since employed to explore synonymous variants. Specifically, we focused on the HIV Tat protein, a transcription factor responsible for activating the long terminal repeat (LTR) promoter and facilitating HIV gene expression in human cells.…”

“…Specifically, we focused on the HIV Tat protein, a transcription factor responsible for activating the long terminal repeat (LTR) promoter and facilitating HIV gene expression in human cells. Using GigaAssays, we previously examined the impact of saturating single-substitution mutagenesis on the activity of the Tat protein [ 18 , 19 ]. We generated a comprehensive library of Tat variants through saturating mutagenesis, encompassing all 1615 possible single amino acid substitutions.…”

“…Our paper is based on a standardized measurement of molecular activities of variants in a single-pot multiplexed assay of 1000s of variants at once. Our proprietary GigaAssay ® has a demonstrated accuracy of ~95%, as assessed with several methods, and thus is suitable for determining the frequency of non-silent synonymous variants [ 18 , 19 ].…”

“…The latter paper adds new important information to the evolving story of non-silent synonymous variants. Our system and design of the approach were advantageous in that it isolates the synonymous variant effect on the direct gene output, drastically reducing the number of potential confounding variables prevalent in examining whole-cell or -organism outcomes such as fitness that are far downstream of its direct function [ 18 , 19 ].…”