Rapid curation of gene disruption collections using Knockout Sudoku

Abstract

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. This method is ∼100× faster and 30× lower in cost than the next comparable method (In-seq) for annotating transposon mutant collections by combinatorial pooling and next-generation sequencing. This method facilitates the rapid, algorithmically guided condensation and curation of the progenitor collection into a high-quality, nonredundant collection that is suitable for rapid genetic screening and gene discovery.

Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF

“…High-throughput pooling and mapping developments allow for simultaneous identification of a large number of Tn mutants that can be traced to specific stocks, and they are faster and less expensive than sequencing individual mutants at this scale ( 51 – 53 ). Recent examples of these are INSeq ( 54 , 55 ), Knockout Sudoku ( 56 , 57 ), and Cartesian pooling-coordinate sequencing (CP-CSeq) ( 58 ). INSeq uses a robotic liquid handling system to create mutant pools.…”

“…Therefore, we consider the “best practice” of arrayed Tn library construction to be manual distribution of Tn mutants into library stock plates. Knockout Sudoku includes predictive algorithms to determine how many colonies must be screened to separate multihit wells as demonstrated by the generation of the authors’ large, arrayed Shewanella oneidensis Tn library ( 56 , 57 ). We did not do large-scale purification of wells with multiple mapped transposon insertions and instead focused on making smaller libraries of high-quality Tn mutants.…”

Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

“…Recent technological advances have vastly lowered the cost and effort required to order random transposon insertion libraries 15,16 . Pooling strategies that combine cultures prior to sequencing and then use computational methods to identify the positions of individual strains can reduce the number of samples that need to be processed by two orders of magnitude 5,7,10,16,17 . The strategy behind various pooling protocols can be separated into two parts.…”

“…Current sequencing approaches for transposon insertion libraries use a flavor of transposon insertion sequencing (Tn-seq/INSeq/ST-PCR). Because these sequencing approaches map transposon insertions for every individual pool, they remain laborious, often requiring multiple processing steps for each individual pool 7,10,15,16 .…”

Identification of a Pathway for Electron Uptake in Shewanella oneidensis

“…To address this, we developed a rapid colorimetric assay to screen all 3,667 members of the S. oneidensis whole genome knockout collection 20,21 and characterize the genetics of EEU. The assay relies upon oxidation of the reduced form of the redox dye anthra(hydro)quinone-2,6-disulfonate (AHDSred for the reduced form and AQDSox for the oxidized form) and is coupled to reduction of the anaerobic terminal electron acceptors fumarate and nitrate [22][23][24] (Fig.…”