bioRxiv2019
NGS studies have uncovered an ever-growing catalog of human variation while leaving an enormous gap between observed variation and experimental characterization of variant function. High-throughput screens powered by NGS have greatly increased the rate of variant functionalization, but the development of comprehensive statistical methods to analyze screen data has lagged behind. In the massively parallel reporter assay (MPRA), short barcodes are counted by sequencing DNA libraries transfected into cells and output RNA in order to simultaneously measure the shifts in transcription induced by thousands of genetic variants. These counts present many statistical challenges, including over-dispersion, depth dependence, and uncertain DNA concentrations. So far, the statistical methods used have been rudimentary, employing transformations on count level data and disregarding experimental and technical structure while failing to quantify uncertainty in the statistical model.\n\nWe have developed an extensive framework for the analysis of NGS functionalization screens available as an R package called malacoda (available from github.com/andrewGhazi/malacoda). Our software implements a probabilistic, fully Bayesian model of screen data. The model uses the negative binomial distribution with gamma priors to model sequencing counts while accounting for effects from input library preparation and sequencing depth. The method leverages the high-throughput nature of the assay to estimate the priors empirically. External annotations such as ENCODE data or DeepSea predictions can also be incorporated to obtain more informative priors - a transformative capability for data integration. The package also includes quality control and utility functions, including automated barcode counting and visualization methods.\n\nTo validate our method, we analyzed several datasets datasets using malacoda and alternative MPRA analysis methods. These data include experiments from the literature, simulated assays, and primary MPRA data. We also used luciferase assays to experimentally validate the strongest hits from our primary data, as well as variants for which the various methods disagree and variants detectable only with the aid of external annotations.\n\nAuthor SummaryGenetic sequencing technology has progressed rapidly in the past two decades. Huge genomic characterization studies have resulted in a massive quantity of background information across the entire genome, including catalogs of observed human variation, gene regulation features, and computational predictions of genomic function. Meanwhile, new types of experiments use the same sequencing technology to simultaneously test the impact of thousands of mutations on gene regulation. While the design of experiments has become increasingly complex, the data analysis methods deployed have remained overly simplistic, often relying on summary measures that discard information. Here we present a statistical framework called for the analysis of massively parallel genomic experiments designed to incorporate prior information in an unbiased way. We validate our method by comparing our method to alternatives on simulated and real datasets, by using different types of assays that provide a similar type of information, and by closely inspecting an example experimental result that only our method detected. We also present the methods accompanying software package which provides and end-to-end pipeline that provides a simple interface for data preparation, analysis, and visualization.