Hi Prasangi,

one could use some RACE-PCR and sequence the results on an NGS machine. trimming the sequence of primers and adaptators and your insert will give you the insertion site sequences to be mapped to the coli genome.

fred

Frederic Lepretre Can you please suggest an article about this method? Thanks

Hatem Zayed Thanks. I will check this

Hi Prasangi,

Another option is ligation-mediated (LM) PCR. This protocol starts with fragmenting the transposon-containing genomic DNA (enzymatically, acoustically, or otherwise) to an average size of ~300bp. Next, double-stranded adapter oligos are ligated to the ends of the fragments (this step may require end-polishing, depending on the method of fragmentation). Two rounds of PCR are conducted with nested primers. One primer binds to the transposon sequence and one binds to the adapter sequence - in this way, you specifically amplify fragments containing transposon ends with adjacent genomic DNA (thus identifying insertion sites). The protocol that I've used includes Illumina primer recognition sequences that allow direct sequencing of secondary PCR products with MySeq, NextSeq, or HiSeq machines.

I have used LM-PCR extensively to identify Sleeping Beauty transposon insertions from a wide range of forward genetic screens. I'm including a link to a BioRxiv pre-print manuscript that utilized our latest version of the LM-PCR protocol, along with the bioinformatic tools to analyze sequencing results. I've also attached a document with more details for the LM-PCR protocol. I'm happy to share/discuss other versions of the protocol we've used if it would be helpful.

https://www.biorxiv.org/content/10.1101/598474v1

Note, the pre-print specifically discusses forward mutagenesis screens in cultured mammalian cells, but the LM-PCR protocol to identify transposon insertion sites should be applicable to most any transposon-based genetic screen.

Best regards,

Jesse