=================================================== Variations =================================================== Variations in transposon preparation --------------------------------------------------- In addition to the the basic mariner and Tn5 methods discussed earlier, there are other variations on this theme that have developed that we will discuss briefly. Phage-based transposition in *Staphylococcus aureus* (2015) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Typically transposons are delivered to recipient cells by conjugation or by transformation. `Santiago et al. `__ adapted a phage-based transposition system to enable high efficiency tranposon delivery in *S. aureus*. Magic pools (2018) ~~~~~~~~~~~~~~~~~~ Instead of using rationale design to tweak the antibiotic resistance and promoter, you can select for constructs that work better. This can be tricky as the plasmid is a suicide plasmid, so how do you know which construct gave you the best results? `This approach, which they term "magic pools" `__, seems to be an elegant way to tackle this issue. .. figure:: images/liu_2017_fig1.jpg :scale: 30 % :align: center :alt: Magic Pools Variations in library preparation --------------------------------------------------- Each method has its own quirks. Even similar methods that rely on MmeI digestion have identified different ways to enrich for the chromosome-transposon junctions required for sequencing. Here are additional methods that extend conceptually beyond INSeq/Tn-Seq library preparation. BarSeq (2015) ~~~~~~~~~~~~~ In the `BarSeq method `__, elements of STM and Tn-Seq are combined for a powerful combination. The library is constructed with each transposon insertion containing a random section of 20 nucleotides (N20). First, the base library is characterized by typical Tn-Seq methods that identify the transposon insertions and also identify the 20mer for each insertion. In subsequent analyses, a simpler library preparation can be conducted (i.e., amplification and sequencing of the 20mer barcodes) to characterize library dynamics. .. figure:: images/wetmore_2015_fig1.jpg :scale: 30 % :align: center :alt: Wetmore 2015 Arrayed mutant libraries --------------------------------------------------- Goodman et al. (2009) ~~~~~~~~~~~~~~~~~~~~~ Typically the transposon libraries from INSeq/Tn-Seq approaches have all of the mutants pooled together. However, for many applications in molecular biology it is very useful to have access to individual mutants. `Goodman et al. `__ devised an ingenious method to accomplish this. Mutants are first arrayed into 96-well microplates. Each mutant is then placed into a specific subset of 24 pools, and those pools are then analyzed by INSeq. The occurrence of a mutant in specific pools uniquely identifies its location in the original library. The only limitation of the method is that arraying the mutants into the pools requires a dedicated epMotion robot to ensure that mutants are faithfully placed in their assigned pools. .. figure:: images/goodman_2009_fig2.jpg :scale: 30 % :align: center :alt: Goodman 2009 Knockout Sodoku (2016) ~~~~~~~~~~~~~~~~~~~~~ A similar combinatorial pooling approach was adapted to be conducted without a detailed robotic algorithm by `Baym et al. `__ in an approach they called "knockout sodoku". Samples are pooled across plates, rows, and columns. The authors used a 96-well multichannel pipet to pool small volumes from the original plates in a specified fashion. .. figure:: images/baym_2016_fig1.jpg :scale: 30 % :align: center :alt: Baym 2016