Rapid Construction of a Whole-genome Transposon Insertion Collection forShewanella oneidensisby 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 nextgeneration 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.

Rapid ordering of barcoded transposon insertion libraries of anaerobic bacteria

“…Our pool design shares the same row and column pools as a previous ST-PCR study, allowing us to compare the performance of the sequencing methods. Top: the log 2 (ratio of row/column reads) should be a Voigt distribution centered around −0.585 8 (red dotted line).The fit Voigt distributions for Bar-seq (blue, this study) and ST-PCR (black, our re-analysis of previous work 8 ) were normalized to an area under the curve of 1 and plotted. Bottom: The fit Voigt distributions (above) were used to predict the likelihood of row and column solutions for all mutant strains that appeared in exactly two row and two column pools.…”

“…We then located barcodes within the ordered library using Bar-seq. We predicted a position for 3517 barcodes within the library, of which 235 were found in more than one position and had to be resolved probabilistically 8 . The final map of transposon insertions in the library identified 3059 wells with a single strain, 350 wells with more than one strain, and 431 wells without a barcode assignment (Figure 7a, top).…”

“…In this protocol, we used barcode sequencing (Bar-seq) to sequence pools and locate mutant strains within the library. Alternative sequencing methods that have been used in previous studies include insertion sequencing (INSeq) 5 , transposon sequencing (Tn-seq) 7 , and semi-random two-step PCR (ST-PCR) 8 . Bar-seq and ST-PCR are significantly simpler and more cost-effective than INSeq and Tn-seq; these PCR-based sequencing methods avoid fragmentation, restriction enzyme digestion, size selection, ligation, and multiple purification steps that are required for INSeq and Tn-seq.…”

“…The pool designs used here and elsewhere 7,8 are optimized for the time and effort required to pool the libraries. If a barcode occurs in exactly one each of the plate, row, and column pools, then for our pool design it has a definite solution for a single location in the library.…”

“…If a strain occurs more than once in the library, then its barcode will appear in more than one of each of the different pool types and, with our pool design, there will be multiple possible solutions for its locations in the library. To predict the correct locations of these repeated transposon insertion strains, a previous study 8 developed a probabilistic solution that we implemented here.…”