Hi Sherine,

In general, the method we use is to collect many hundreds of thousands of transposon mutants together in mini pools then sequence them. That data cen be used to define essential genes, as like you said, the genes/operons that are not hit by the transposon are essential. You can be further selection by growing the mini pools in different conditions (nutrients, antibiotics, etc) and then sequencing to define conditionally essential genes.

I dont think there is a review yet on all the methods used to analyse TnSeq data, but some methods define essentiality by read count per gene, others take into account gene length, others use Monte Carlo computational methods to determine statistical significance of genes that are hit vs random transposon placement.

You can use software like TnSeq explorer, or ESSENTIALS, or the scripts provided in GitHub by individual papers. Below is a publication from my lab where we do this:

"Comparative analysis of the Burkholderia cenocepacia K56-2 essential genome reveals cell envelope functions that are uniquely required for survival in species of the genus Burkholderia"

AND

"Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ"

Others references:

"Genome-Scale Identification of Resistance Functions in Pseudomonas

aeruginosa Using Tn-seq"

AND

"ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data"

Hope this helps!

Andrew