Transposon insertion sequencing (Tn-Seq) is an NGS method to en masse map genomic locations of transposon insertions in a pool of mutants. Insertion mutants are usually made using synthetic mini transposons like Tn5 and Himar (in bacteria), Sleeping Beauty & Piggybac (in mammalian cells), and others like Mos (in Drosophila \/ C. elegans).There are several NGS methods to perform de novo Tn-Seq mapping of a library of mutants. Some of the key ones are listed below. Van Opijnen, Tim, Kip L. Bodi, and Andrew Camilli. "Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms."Nature methods 6.10 (2009): 767. - Uses MmeI (a type IIs restriction enzyme) digestion to generate ~ 18bp genomic DNA overhangs from Mariner transposon insertions. Adapters are ligated to the fragments, the transposon - genome junction is enriched by PCR before being sequenced. Because of the short genomic sequence available for alignment to reference, mapping accuracy can be lower compared to fragmentation based library prep methods.Langridge, Gemma C., et al. "Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants."Genome research (2009). - DNA extracted from a pool of mutants is sheared\/fragmented to a desired fragment size (usually 300 - 400 bp), fragments are end-repaired, and the transposon - genome junction is enriched by PCR before being sequenced.The list is only intended to highlight two major approaches to Tn-seq library prep - restriction digestion based or fragmentation based. There are several variations on each approach that are now available for the discerning scientist. There are also less known but interesting alternative methods like HTML-PCR.After trying out several of the above methods, we have developed a Tn-Seq library preparation protocol that incorporates desirable features from existing protocols and new features that will improve data quality, while being easy to perform (an undergraduate student with some wet-lab experience can go through the protocol in a day). The protocol is conceptually similar to the TraDIS protocol (see the Langridge et al paper above).Key features are:1. Uses the NEB Ultra II FS kit for enzymatic fragmentation of genomic DNA, end-repair of fragments, and ligation of adaptor. The kit allows performing all the reactions sequentially in a single tube (one-pot prep).2. Incorporates Unique Molecular Identifiers (UMI) to enable removing PCR duplicates from sequencing data. Improves read count accuracy when the data is to be used for read-frequency based analyses (fitness or log fold change).3. Includes 'best practices' library features like color diversity phasing and separate indexing step to save cost on adaptor oligos.The method is also automation friendly.