Search found 211 matches

by cshaffer
Thu Mar 12, 2009 2:39 am
Forum: Sequence Improvement Questions
Topic: Contig SIze
Replies: 3
Views: 9504

size errors on large fragments

the version of the genome browser we have on gander now includes the positions of the fosmids. go to http://gander.wustl.edu click on genome browser and select the right species. To see where this clone sits with respect to the whole genome shotgun assembly enter this location: scaffold_14822:502,67...
by cshaffer
Tue Mar 10, 2009 6:45 pm
Forum: Sequence Improvement Questions
Topic: Contig SIze
Replies: 3
Views: 9504

that is a small fosmid

That is really a small insert size for a fosmid. They do come that small sometimes but rarely. I think the size difference is mostly since the digest will include the size of the vector and the assembly view does not. I believe the size of the vector in these fosmids is about 8kb. So this would be a...
by cshaffer
Mon Mar 02, 2009 1:17 am
Forum: Sequence Improvement Questions
Topic: Tandem Repeats?
Replies: 4
Views: 11668

reads that go too far

Don't worry to much about the ends. We have a series of overlapping fosmids (fosmids overlap by at least 2 kb, in regions where picking was poor the overlap might be as high as 10 - 15 kb) so if bases are low quality at one end they will be covered by the neighbor. When we put everything together we...
by cshaffer
Fri Feb 27, 2009 5:50 pm
Forum: Sequence Improvement Questions
Topic: tagging problems
Replies: 2
Views: 8205

changing tag size

yes sounds like a computer glitch. But try this to make it easier: 1. create the tag on the first base of where you want the tag. 2. once created right click on the tag to get a menu and select the "Tag: blah blah blah show more info?" 3. this will open a window with more info on the tag including t...
by cshaffer
Fri Feb 27, 2009 1:55 am
Forum: Sequence Improvement Questions
Topic: Tandem Repeats?
Replies: 4
Views: 11668

consed problem

I have seen consed do something like this but never this bad. I have seen what you are showing me in a simpler version where consed will put a gap at different places in different reads and end up creating extra bases in the consensus. When considering only the high quality reads the alignment looks...
by cshaffer
Wed Feb 25, 2009 6:11 pm
Forum: Sequence Improvement Questions
Topic: Problems with new reads
Replies: 3
Views: 10606

chemistry choices

Yes, the only reason for the choice of reactions is efficiency. Everything has to be run in batches of 96. The first year we let everyone choose any combination but then we would get very few oligos in each class. I would then have to bin together the oligos to make whole plates of 96. Since I never...
by cshaffer
Tue Feb 24, 2009 8:35 pm
Forum: Sequence Improvement Questions
Topic: restriction maps:degree of acceptable diff (real v in-silico
Replies: 3
Views: 9544

systematic error

Ok, I am grading the final reports and have now seen this same issue (systematic smaller in silico bands) in 2 of 4 reports. Curiously they were both the SacI digests. This gives me more confidence that your digest does indeed confirm your assembly. Also it looks like these gels were a little less s...
by cshaffer
Mon Feb 23, 2009 7:53 pm
Forum: Sequence Improvement Questions
Topic: changing trace position changes peak height?!
Replies: 1
Views: 6225

consed will automatically adjust the vertical scale

Yes, when you scroll the the right consed is changing the vertical scale to show you that large T peak. This scaling reduces the "real" peaks to the point of being almost a flat line. This is a common problem with Dye blobs and how they interfere with everything. You can get the "real" peaks back by...
by cshaffer
Mon Feb 23, 2009 7:52 pm
Forum: Sequence Improvement Questions
Topic: low-quality base calls, but high-quality trace peaks?
Replies: 3
Views: 10649

recalling reads

The short answer is yes you have to do it manually. There are tricks that would use more modern basecallers that now know to ignore the dye blobs. These basecallers would probably call this read just fine, unfortunately these basecallers can not easily be switched in for phred. I am not even sure ex...
by cshaffer
Mon Feb 23, 2009 7:19 pm
Forum: Sequence Improvement Questions
Topic: restriction maps:degree of acceptable diff (real v in-silico
Replies: 3
Views: 9544

digests

For those that are not aware, the info for interpretation of digests is outlined in a document found on the main GEP page: http://gep.wustl.edu/curriculum/course_materials/finishing/RestrictionDigests.pdf As this document states all in silico bands between 1kb and 10 kb should be within 2% of the si...
by cshaffer
Thu Feb 19, 2009 5:04 pm
Forum: Sequence Improvement Questions
Topic: restriction map:'real' frags total ~ 100kb
Replies: 1
Views: 6339

digests

I am guessing the digest did not really work and the bands you are seeing are partials. We see this from time to time. Are there any other digests you can use to confirm? this one looks like a total loss to me. If you cannot get 2 restriction digests for confirmation then this should be noted in the...
by cshaffer
Thu Feb 19, 2009 3:33 am
Forum: Sequence Improvement Questions
Topic: low-quality base calls, but high-quality trace peaks?
Replies: 3
Views: 10649

phred basecalls

I have seen things like this happen when there is a dye blob in the read. This can sometimes throw off all the basecalls for the whole read. It sure looks like high quality to me. I always believe my eyes before I believe phrap :) At least when they contradict each other. So, yes, go ahead and edit ...
by cshaffer
Thu Feb 19, 2009 3:27 am
Forum: Sequence Improvement Questions
Topic: trashing garbage reads?
Replies: 7
Views: 16338

removing PHD files

yes you cannot just remove the PHD files as the first thing phredphrap does is compare the contents of the chromat_dir and phd_dir. Any files in the chromat_dir and not in the phd_dir are assumed to be new data so phred does the basecalling to create a new phd file before calling phrap to do the ass...
by cshaffer
Sat Feb 14, 2009 3:42 pm
Forum: Sequence Improvement Questions
Topic: trashing garbage reads?
Replies: 7
Views: 16338

deleting reads

Yes you could delete files from the chromat_dir, if you do that you should also delete the *.phd file from the phd_dir as phrap assembles all the reads in the phd_dir it does not care what is in the chromat_dir. Of course if you go back to an older ace file the read will still be there and if you tr...
by cshaffer
Fri Feb 13, 2009 3:53 pm
Forum: Sequence Improvement Questions
Topic: trashing garbage reads?
Replies: 7
Views: 16338

junk reads

yes you can pull the read out entirely. This will work but if you rerun phredphrap it will probably stick that read right back in. And then you can pull it out again. So if you go this route you might want to use the "add new reads" to add new data instead of running phredphrap. Another option that ...