Strange Vector Problems
Posted 28 September 2011 - 02:28 PM
I have cloned environmental DNA into a Fosmid vector, which is essentially an 8.1 kb plasmid under inducible copy number control using L-Arabinose. I am using Sanger Sequencing to sequence about 800 bases of the apprx. 40 kb insert. Most of my sequences are fine, however when I use BLASTn about 10% of the sequences come back as vector, even after vector trimming.
What is even stranger is that the vector that shows up on the BLAST is not the vector I used. Typically about the first 300 bases are shown to be vector in BLAST (and they are the same 300 for each case), and if I remove them manually and BLAST the remaining 500 bases, I get a legitimate insert. Also, each clone can be sequenced from each end of the insert, and this problem is usually present only one side (but no pattern in terms of forward or reverse primer). When I take the 300 or so bases that are "vector" from the BLAST, and search my vector sequence for it, it is not present even with 25 mismatches.
There are other cases where some sequences have a different vector than the two mentioned above, and it is only about 80 bases when examined with BLAST. No other results show up, but if I cut this vector (which is not my vector because I have already clipped it and the sequence has no homology to the vector I am using) and BLAST the rest of the sequence a legitimate insert is detected through BLAST.
Any ideas on what could be cuasing this? Mispriming during sequencing? software limitations?
Posted 29 September 2011 - 12:49 PM
Best of Luck.
Posted 29 September 2011 - 01:24 PM
The vectors that come up on the BLASTn searches are not being used in the lab, and the inserts were prepared through mechanical shearing, so no restricion enzymes. The "extra vector" that persists after trimming and is not the vector used in construction is the same for each clone, and is consistently about 294-300 bases. Whats strange is that for example the forward direction may have this problem and the reverse wont. In some cases I have resequenced the clone and the vector is not there. Could this be a problem with sequencing since is it the same vector sequence for each clone, and resequencing has some effect. However, I mention again that this vector is not the vector that I or anyone in my lab uses, so it is puzzling why it is there, and interesting that clipping it off manually seems to give an appropriate insert.
Posted 30 September 2011 - 05:08 AM
Best of Luck.