Dataset Information

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

ABSTRACT:

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Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology.

Availability and implementation

The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-Tradis

Contact

parkhill@sanger.ac.uk

Supplementary information

Supplementary data are available at Bioinformatics online.

SUBMITTER: Barquist L

PROVIDER: S-EPMC4896371 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Publications

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

Barquist Lars L Mayho Matthew M Cummins Carla C Cain Amy K AK Boinett Christine J CJ Page Andrew J AJ Langridge Gemma C GC Quail Michael A MA Keane Jacqueline A JA Parkhill Julian J

Bioinformatics (Oxford, England) 20160121 7

<h4>Unlabelled</h4>Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an opt ...[more]

PMID: 26794317

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Project description:MotivationProbabilistic Identification of bacterial essential genes using transposon-directed insertion-site sequencing (TraDIS) data based on Tn5 libraries has received relatively little attention in the literature; most methods are designed for mariner transposon insertions. Analysis of Tn5 transposon-based genomic data is challenging due to the high insertion density and genomic resolution. We present a novel probabilistic Bayesian approach for classifying bacterial essential genes using transposon insertion density derived from transposon insertion sequencing data. We implement a Markov chain Monte Carlo sampling procedure to estimate the posterior probability that any given gene is essential. We implement a Bayesian decision theory approach to selecting essential genes. We assess the effectiveness of our approach via analysis of both simulated data and three previously published Escherichia coli, Salmonella Typhimurium and Staphylococcus aureus datasets. These three bacteria have relatively well characterized essential genes which allows us to test our classification procedure using receiver operating characteristic curves and area under the curves. We compare the classification performance with that of Bio-Tradis, a standard tool for bacterial gene classification.ResultsOur method is able to classify genes in the three datasets with areas under the curves between 0.967 and 0.983. Our simulated synthetic datasets show that both the number of insertions and the extent to which insertions are tolerated in the distal regions of essential genes are both important in determining classification accuracy. Importantly our method gives the user the option of classifying essential genes based on the user-supplied costs of false discovery and false non-discovery.Availability and implementationAn R package that implements the method presented in this paper is available for download from https://github.com/Kevin-walters/insdens.Supplementary informationSupplementary data are available at Bioinformatics online.

Dataset Information

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

Unlabelled

Availability and implementation

Contact

Supplementary information

Publications

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries.

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