Library Plates:: Sequencing Templates Production

Plasmids were prepared by ligating pieces of genomic DNA generated from a double-digest using BamHI and BglII. The DNA concentration was low to insure that most ligation was "self-ligation" of the ends of a single piece of linear DNA. Circularization occurs at the cohesive overhangs generated by the two enzymes. Sequencing from the right end of RescueMu up to the ligation site yields the genomic DNA that we know was next to the transposon in vivo. Sequence beyond the ligation site most likely comes from the "left" side of the RescueMu transposon in vivo. Thus sequencing through the ligation site generates sequence that comes from two different places in the genome and is in two different directions. The sequences before and after a predicted ligation site were therefore separated and submitted under their own identification (ID) numbers to the GSS database at Genbank.

Below is a detailed explanation of the situation.

The recognition site for BamHI is

and the recognition site for BglII is

Same Enzyme Ligation: If the ligation joined two ends where both were cut by BglII or both were cut by BamHI, the restriction site would have been recreated. Finding one of the two restrictions sites during sequencing is therefore a good indication that we have found the ligation site. There is a slight chance for some restriction sites to be missed during the double-digest so this is not a 100 percent sure prediction.

Mixed Site Ligation: If the ligation joined two ends where one was cut by BglII and the other by BamHI, then we would end up with one of the two junction ligation sequences below:

Because these ligation sequences are not restriction sites, they could have been present in the genomic DNA (assuming a random distribution, every 4⁶ bases) and therefore not represent the true ligation site.

If a possible ligation site (a BamHI or BglII restriction site or either of the two junction ligation sites) was found during sequencing, the sequence before the ligation site was submitted as normal with its plate id, well number, direction, and run number. The sequence after the ligation site, if it was at least 30 bases, was submitted with an ID based on the original sequence ID as follows:

Ligation Site Type	Original Sequence ID	Post-ligation Sequence ID
1 enzyme both ends	1006001A01.x1	1006001A01.1EL_x1
2 enzymes	1006001A01.x1	1006001A01.2EL_x1

In this way, the post-ligation sequence is tied back to the original sequence ID and indicates the ligation type. The comment field in the Genbank record describes what was done for each sequence, too.