consed and restriction digests

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cshaffer
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consed and restriction digests

Post by cshaffer » Mon Mar 03, 2008 5:30 pm

When looking at the digests you and your students may have noticed a large number of mis-matching bands. This is probably not due to a mis-assembly but instead due to consed failing to link the insert and vector in silico prior to digestion. This has the consequence that the in silico digests has two extra cut sites that are not really in the fosmid. To complicate matters even more there are some sites very close to the vector/insert boundary so these digests will look just fine for some enzymes and not others even though there is that added in silico cut site.

There is one thing you can do that might help, consed does a better job of joining insert to vector if you select "entire single contig" instead of the default "Entire clone" in top right of the "Select Emzymes and Contigs" window. After you select "entire single contig" enter the number of the contig that is your insert. Select your enzymes as you normally would and click OK.

You should see an error message something like this:

Code: Select all

"can't determine which end of vector is connected to the right end of contig Contig1 so you might need to complement the vector" 
This is good because it means consed is trying to join the insert with the vector but it cannot find enough sequence overlap to know which end of the insert goes with which end of the vector. So it is warning you that consed is just going to guess.

If consed guesses correctly then your map should be correct. If consed guesses incorrectly then you will have two fragments that do not match, each one a fragment with one end in the insert and one end in the vector. If this happens click "compl vector" this will flip the orientation of the vector with the insert and then everything should match.

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