36
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Sort-seq under the hood: implications of design choices on large-scale characterization of sequence-function relations

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Background

          Sort-seq is an effective approach for simultaneous activity measurements in a large-scale library, combining flow cytometry, deep sequencing, and statistical inference. Such assays enable the characterization of functional landscapes at unprecedented scale for a wide-reaching array of biological molecules and functionalities in vivo. Applications of sort-seq range from footprinting to establishing quantitative models of biological systems and rational design of synthetic genetic elements. Nearly as diverse are implementations of this technique, reflecting key design choices with extensive impact on the scope and accuracy the results. Yet how to make these choices remains unclear. Here we investigate the effects of alternative sort-seq designs and inference methods on the information output using mathematical formulation and simulations.

          Results

          We identify key intrinsic properties of any system of interest with practical implications for sort-seq assays, depending on the experimental goals. The fluorescence range and cell-to-cell variability specify the number of sorted populations needed for quantitative measurements that are precise and unbiased. These factors also indicate cases where an enrichment-based approach that uses a single sorted population can offer satisfactory results. These predications of our model are corroborated using re-analysis of published data. We explore implications of these results for quantitative modeling and library design.

          Conclusions

          Sort-seq assays can be streamlined by reducing the number of sorted populations, saving considerable resources. Simple preliminary experiments can guide optimal experiment design, minimizing cost while maintaining the maximal information output and avoiding latent biases. These insights can facilitate future applications of this highly adaptable technique.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12864-016-2533-5) contains supplementary material, which is available to authorized users.

          Related collections

          Most cited references38

          • Record: found
          • Abstract: found
          • Article: not found

          Bacterial persistence as a phenotypic switch.

          A fraction of a genetically homogeneous microbial population may survive exposure to stress such as antibiotic treatment. Unlike resistant mutants, cells regrown from such persistent bacteria remain sensitive to the antibiotic. We investigated the persistence of single cells of Escherichia coli with the use of microfluidic devices. Persistence was linked to preexisting heterogeneity in bacterial populations because phenotypic switching occurred between normally growing cells and persister cells having reduced growth rates. Quantitative measurements led to a simple mathematical description of the persistence switch. Inherent heterogeneity of bacterial populations may be important in adaptation to fluctuating environments and in the persistence of bacterial infections.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Phenotypic diversity, population growth, and information in fluctuating environments.

            Organisms in fluctuating environments must constantly adapt their behavior to survive. In clonal populations, this may be achieved through sensing followed by response or through the generation of diversity by stochastic phenotype switching. Here we show that stochastic switching can be favored over sensing when the environment changes infrequently. The optimal switching rates then mimic the statistics of environmental changes. We derive a relation between the long-term growth rate of the organism and the information available about its fluctuating environment.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Deep mutational scanning: a new style of protein science.

              Mutagenesis provides insight into proteins, but only recently have assays that couple genotype to phenotype been used to assess the activities of as many as 1 million mutant versions of a protein in a single experiment. This approach-'deep mutational scanning'-yields large-scale data sets that can reveal intrinsic protein properties, protein behavior within cells and the consequences of human genetic variation. Deep mutational scanning is transforming the study of proteins, but many challenges must be tackled for it to fulfill its promise.
                Bookmark

                Author and article information

                Contributors
                elevine@fas.harvard.edu
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central (London )
                1471-2164
                9 March 2016
                9 March 2016
                2016
                : 17
                : 206
                Affiliations
                Department of Physics and FAS Center for Systems Biology, Harvard University, 17 Oxford St., Cambridge, MA USA
                Article
                2533
                10.1186/s12864-016-2533-5
                4784318
                26956374
                06d8e25a-4913-46aa-8ce1-1b0da5bc6876
                © Peterman and Levine. 2016

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 6 November 2015
                : 25 February 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation (US);
                Award ID: PHY-1205494
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation (US);
                Award ID: PHY-1219334
                Award Recipient :
                Categories
                Methodology Article
                Custom metadata
                © The Author(s) 2016

                Genetics
                sequence-function relations,systems biology,high-throughput sequencing,fluorescence-activated cell sorting

                Comments

                Comment on this article