Transposon Sequencing (2009)ΒΆ
At least four different groups developed technology to identify transposon insertion sites via short-read sequencing. All of the technologies are widely adaptable to various organisms. There are differences in the type of tranpsoson used (mariner/Tn5) and method to enrich the transposon-chromsome junctions, but generally break down into two strategies (from van Opijnen and Camilli 2003)
Key references for INSeq, Tn-Seq etc.:
| Discovery | Notes |
|---|---|
| Goodman et al. 2009 | INSeq (MmeI-based) |
| van Opijnen and Camilli 2009 | Tn-Seq (MmeI-based) |
| Gawronski et al. 2009 | HITS (MmeI-independent) |
| Langridge et al. 2009 | TraDIS (MmeI-independent) |
| Gallagher et al. 2011 | Circularization, RE-dependent |
| Review Articles | Notes |
|---|---|
| van Opijnen and Camilli 2003 | Broad overview |
| Chao et al. 2016 | Analysis of dense libraries, bottlenecks |
| Kwon et al. 2016 | Studies using transposon sequencing |
| Methods | Notes |
|---|---|
| Goodman et al. 2011 | Bead-based, detailed INSeq protocol. |
For this workshop, we will focus on the Goodman & Gordon INSeq method, which is similar conceptually to the van Opijnen & Camilli Tn-Seq method.
The INSeq library preparation method was first described in 2009 and was refined in 2011 to use a biotinylated linear amplification step. Subsequent molecular steps occur with the sample immobilized on a magnetic streptavidin bead, which enables reactions to be performed in a multiplexed fashion with a low volume of reagents.
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