Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing.

Evelyn Plets,Kristof Vandewalle,Nico Callewaert,Marnik Vuylsteke,Yvan Saeys,Nele Festjens

doi:10.1038/ncomms8106

Abstract

Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants. Alternatively, where these methods are lacking, the construction of pre-characterized libraries of mutants can be extremely valuable. However, this can be complex, expensive and time consuming. Here, we describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections. We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The method is applicable to any entity for which sequence-tagged identification is possible.

Highlights

Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants
An example can be found in tuberculosis research, where mutants in genes of interest are among others required for the study of potential drug targets and for live attenuated vaccine engineering[4,5]
Ordered Tn insertion libraries can be picked from such Tn mutant mixtures and PCR-screened for insertions in a locus of interest[9], but this procedure needs to be repeated for every gene and is prone to failure due to the high GC content of these genomes

Summary

Introduction

Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants. We describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. We have aimed at developing an easy-to-implement, cost- and time-effective, massively parallel sequencing-based approach (called Cartesian Pooling-Coordinate Sequencing or CP-CSeq) to deal with the characterization of such large collections of sequence-tagged clones We illustrate it here for a large 9,216 clone ordered Tn insertion mutant library of the vaccine strain M. bovis Bacillus Calmette–Guerin (BCG). We demonstrate how this approach strongly contributes towards the endeavour of generating a mutant for every gene in the genome of such slow-growing Mycobacteria, which constitute some of the most important pathogens of mankind

Methods

Results

Conclusion