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Next-generation sequencing Abstract Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, Amplicon PC24 construction has required an extraordinary technical effort.

Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing Amplicon PC24 in a single day, a to fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation Amplicon PC24 a minimal representative set of mutants, validation, and curation.

Starting from a progenitor collection of 39, mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3, genes that is functionally validated by high-throughput kinetic measurements of quinone reduction. Introduction Shewanella oneidensis MR-1 is one of the archetypal members of the Amplicon PC24 microbes 1. Members of this class are capable of transferring electrons to, from, or between metabolism and a remarkable variety of external substrates, ranging Amplicon PC24 metal ions 2 to solid surfaces including electrodes 1. The Shewanellae serve as model organisms for understanding the role of microbes in the biogeochemical cycling of metals 3 and carbon 4 as well as the geochemical evolution of the Earth 56.

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This cytochrome network provides S. However, despite intense research, the functions of over half of its genes remain unknown 13 and fundamental discoveries Amplicon PC24 its nature continue to be made 12 Although the centrality of the MtrABC complex in EET is well established 15Amplicon PC24 continues about the full complement of proteins used in this process, particularly in electron transfer from the inner membrane, across the periplasm, to the outer membrane 161718 There remains an ongoing need for the development of new, highly accessible tools for the genetic manipulation and characterization of S.

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Aside from the genome sequence, a single-gene knockout collection is one of the most valuable genetic tools for any organism. Transposon mutagenesis, in which a defined DNA sequence is randomly integrated into a single genomic site, allows the straightforward creation of extremely large gene disruption mutant libraries for a wide variety of microorganisms 21 Recently developed techniques that use next-generation sequencing NGS of pooled transposon mutant libraries 232425 have made dramatic advances in the characterization of genes that can be selected for fitness contributions 26 to species including S. Nevertheless, clonally isolated collections of mutants remain critically important for the characterization of phenotypes such as virulence factors 2930secondary and cryptic metabolite production 31 and behaviours synonymous with Shewanellae, such as biofilm formation 3233 and EET Conventionally, a small collection 5,—8, members of transposon mutants is constructed solely for a specific genetic screen, hits are identified and isolated while the rest of the collection is often discarded.

Additionally, these collections do not facilitate testing of specific gene function predictions Amplicon PC24 The utility of comprehensive, non-redundant, catalogued curated collections is hard to overstate: While curated collections made by targeted gene deletion remain the gold standard 37their construction requires an extraordinary technical and financial undertaking, and as a result, only a handful exist 233538394041 Supplementary Note 1.

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Cataloging extremely large transposon mutant collections by Sanger sequencing and condensation 304243 significantly reduces costs, but is still prohibitive Supplementary Amplicon PC24 1. NGS has led to an explosion of genomic data in the past decade.

This has inspired the development of techniques that dramatically reduce the cost of cataloging transposon mutant collections by combinatorial pooling, followed by a single-NGS experiment, and analysis of the resulting data set 24Amplicon PC24 The problem solved in this analysis, reconstructing the location of genomic coordinates on a grid of wells, resembles the number placement puzzle Sudoku This exciting development has facilitated the construction Amplicon PC24 a growing number of curated collections for pathogens and pathogen surrogates 3645 However, the costs, complexity and low speeds of the robotic hardware needed to combine samples before sequencing make these methods inaccessible to many investigators Supplementary Note 1. This limitation has inspired schemes that do not rely on robotics for sample preparation However, the data sets produced by these manual pooling methods pose significant challenges for reconstruction due to the presence of non-unique sequences that map back to both their originating locations and to many other artifactual ones Here, we describe a low-cost, easily implemented, rapid and generalizable method for automatically cataloging extremely large transposon mutant collections and then guiding the construction and validation of a condensed, quality-controlled QC collection.

We have developed an algorithm that uses self-consistency within a sequencing data set to disambiguate the location of non-unique transposon mutants. This allows the use of an extremely rapid, simple manual combinatorial pooling method that is able to pool a Amplicon PC24, member progenitor S.

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