Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku

Abstract

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their 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 library in a single day, a 30- to 100-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 to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

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.…”

Chemical genetics in drug discovery

“…In the past decade mutant libraries have been constructed in a plethora of bacteria and fungi [7] . More recently, our proficiency in generating genome-wide pooled mutant libraries [8] and de-convoluting via multiplexing sequencing approaches 9 , 10 has brought us to a stage where libraries can be created for almost any microorganism [11] . Although natural genetic variation is frequently used in chemical genetics in human cell lines ∗5 , ∗6 , ∗12 , this unlimited resource has only been recently explored in bacteria [13] , leading to similar abilities to delineate drug function as ordered libraries.…”

A Novel Computational Approach for Identifying Essential Proteins From Multiplex Biological Networks

“…The identification of essential proteins can help us to understand the basic requirements of living organisms, and it can also play an important role in drug design (Dubach et al, 2017), genetic disease diagnosis (Zeng et al, 2017), and drug synergy prediction in cancers (Li et al, 2018). Traditional experimental approaches, such as gene knockouts (Narasimhan et al, 2016), RNA interference (Inouye, 2016), and Knockout Sudoku (Baym et al, 2016), are time-consuming and costly. Over the last few decades, high-throughput technologies have produced a tremendous amount of protein interaction network (PIN) data that provide us with new opportunities to detect essential proteins through the use of computational approaches.…”