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Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments


The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical–genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. The software has also successfully processed data from Schizosaccharomyces pombe, Escherichia coli, and Zymomonas mobilis mutant collections. We provide general recommendations for the design of large-scale, multiplexed barcode sequencing experiments. The procedure outlined here was used to score interactions for ~4 million chemical-by-mutant combinations in our recently published chemical–genetic interaction screen of nearly 14,000 chemical compounds across seven diverse compound collections. Here we selected a representative subset of these data on which to demonstrate our analysis pipeline. BEAN-counter is open source, written in Python, and freely available for academic use. Users should be proficient at the command line; advanced users who wish to analyze larger datasets with hundreds or more conditions should also be familiar with concepts in analysis of high-throughput biological data. BEAN-counter encapsulates the knowledge we have accumulated from, and successfully applied to, our multiplexed, pooled barcode sequencing experiments. This protocol will be useful to those interested in generating their own high-dimensional, quantitative characterizations of gene or chemical function in a high-throughput manner.

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Fig. 1: Overview of multiplexed barcode sequencing experiments and their processing using the BEAN-counter software.
Fig. 2: Design of large-scale, pooled and multiplexed chemical–genetic interaction screens.
Fig. 3: Schematic of the steps involved in processing large-scale interaction screens using BEAN-counter.
Fig. 4: The large signature that we observe in and remove from most of our datasets.
Fig. 5: An inoculation date-related effect in one of our datasets.
Fig. 6: Typical barcode and index tag abundance distributions.
Fig. 7: Manual examination of the dataset to flag mutants and conditions for removal.
Fig. 8: Analysis of same-compound, same-index-tag, and same-lane correlations to detect the presence of batch effects and uninformative signal.
Fig. 9: Removal of large, uninformative signature via singular-value decomposition (SVD).

Code availability

The source code for BEAN-counter is available from It requires a license for use (the license can be obtained at It is free for academic use and can be purchased on a per-project basis for commercial use.

Data availability

All data needed to process the example dataset into chemical–genetic interaction scores are available at These data are a subset of the complete large-scale chemical–genetic interaction dataset, which is available from, the supplementary material of the associated article10, or the corresponding author upon reasonable request.


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S.W.S. thanks B. VanderSluis for proofreading the manuscript and testing the software and also A. Becker at the University of Minnesota Genomics Center for discussions regarding amplicon sequencing issues. This work was supported by RIKEN ( Strategic Programs for R&D, the National Institutes of Health (; R01HG005084, R01GM104975), and the National Science Foundation (; DBI 0953881). S.W.S. was supported by an NSF Graduate Research Fellowship (00039202), an NIH Biotechnology training grant (T32GM008347), and a one-year fellowship from the University of Minnesota Bioinformatics and Computational Biology (BICB) Graduate Program ( S.C.L. and J.S.P. were supported by a RIKEN Foreign Postdoctoral Research Fellowship. S.C.L. was supported by a RIKEN CSRS ( Research Topics for Cooperative Projects Award (201601100228) and a RIKEN FY2017 Incentive Research Projects Grant. H.N.W. was supported by a one-year BICB fellowship from the University of Minnesota. C.B. was supported by JSPS KAKENHI ( grant no. 15H04483. C.L.M. and C.B. are fellows in the Canadian Institute for Advanced Research (CIFAR, Genetic Networks Program. Computing resources and data storage services were partially provided by the Minnesota Supercomputing Institute and the UMN Office of Information Technology, respectively. Software licensing services were provided by the UMN Office for Technology Commercialization. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations



C.B., M.Y., C.L.M., J.S.P., and S.C.L. conceived the project. R.D. and C.L.M. designed and R.D. wrote the original analysis software, which was extended and re-implemented as a full analysis pipeline by S.W.S. with assistance from J.N. and H.N.W. S.C.L., J.S.P., and Y.Y. generated chemical–genetic interaction data for software development and testing. J.N., S.C.L., and J.S.P. performed extensive user testing of the software. H.O. provided the RIKEN Natural Product Depository compounds used in the example dataset. S.W.S. wrote the manuscript with input and editing from all authors.

Corresponding author

Correspondence to Chad L. Myers.

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Competing interests

The authors declare competing financial interests. A license is required to use the BEAN-counter software ( It is free for academic use and must be purchased on a per-project basis for commercial use.

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Key references using this protocol

Piotrowski, J. S. et al. Nat. Chem. Biol. 13, 982–993 (2017):

Morales, E. H. et al.. Nat. Commun. 8, 15320 (2017):

Integrated supplementary information

Supplementary Figure 1 The BEAN-counter configuration file provides necessary information for the pipeline.

Schematic showing how the configuration file coordinates the processing of pooled interaction screening data by specifying the location of the raw data, the structure of the PCR amplicons, and the mappings from genetic barcode to mutant and index tag to condition. Columns in bold are required in order to process data with BEAN-counter.

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Simpkins, S.W., Deshpande, R., Nelson, J. et al. Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments. Nat Protoc 14, 415–440 (2019).

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