AT repeats

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drevie
Posts: 67
Joined: Sun Feb 04, 2007 10:23 pm
Location: California Lutheran University, Thousand Oaks, CA

AT repeats

Post by drevie » Tue Mar 27, 2007 9:42 pm

Now that we are almost finished finishing, it appears that almost all of the difficult problems were due to AT repeats. One project has an AT repeat region, about 10 bp, then a second AT repeat region. They haven't been able to get a good sequence for those 10 bp. Another group can't close a region that seems to have an AT repeat, 100-200 bp, then another AT repeat. In both cases, they have tried >1 set of primers from each direction using dGTP and 4:1 chemistries. Today they tried again, so it is still possible that they will get the regions to work, but it seems unlikely.

My guess is that some other method needs to be used, such as higher temp or modifications to the chemistries. Is this what the prof. finishers finally resort to in these cases? Or are we just lame? :?

cshaffer
Posts: 211
Joined: Sun Feb 04, 2007 10:29 pm
Location: Washington University in St Louis
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sequence killers

Post by cshaffer » Tue Mar 27, 2007 10:28 pm

Many ssr (simple sequence repeats) will kill a reaction. Thus you will never get reads to go through and give you uniq sequence on the other side.

For your 10 bp of low quality with AT on each side such that no high quality reads get to the 10 bp, now something like this is dealt with depends on the finishing standards set for the project. For mouse standard (which is our goal) the last way in which a region can pass is if it is "reviewed by an experienced finisher". Depending on how horrible the traces look this might get passed on without more reactions, and yes there is some data of this quality in some of the published genomes.

For the region is 100-200 bases between AT's can you get any hints of data to call a primer by hand? If you can get a primer to anneal in between the two you could cover the internal region with a pair of primers. If you want to try this there are some special techniques you need to follow when you upload your ace file for ordering oligos. Let me know if you want to try.

If these final reactions do not help simply tag the region using either a comment tag or the "data needed" tag and include a note in the presubmit word document, put that document in the edit_dir and submit the project. Wilson and I will then collect all of these problem regions and have them reviewed by a professional finisher.

As a final note there are other labor intensive ways to get these sequences if you really really want them. The most common is to make a shatter library in which a piece of DNA is broken down in to 20-50 base fragments, these fragments are shotgun cloned into a sequencing vector and then a bunch of these clones are sequenced. If you are lucky you will get a few fragments that have just the right end to give you the data you need.

drevie
Posts: 67
Joined: Sun Feb 04, 2007 10:23 pm
Location: California Lutheran University, Thousand Oaks, CA

Post by drevie » Wed Mar 28, 2007 3:45 pm

The 10 bp between two AT repeats has about half the bases high quality, so it should be resolvable by an expert.

I can't look more closely at the 100-200 bp region between the two AT repeats, as we mailed the computer to Wilson to fix our phrap problem. Otherwise I'd try making primers.

Right now one end has a high quality AT repeat, then 100 bp low quality, then low quality AT repeats. The other end has the same pattern except that there is about 200 bp between the AT repeats. We discovered this while trying to manually join the regions. It's too bad I didn't notice this earlier and we could have tried to make primers in the low-quality regions.

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