Starting out from fosmid ends

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cjones
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Starting out from fosmid ends

Post by cjones » Tue Feb 05, 2013 2:48 am

I do jigsaw puzzles from the outside in, starting with the edge pieces. Is there some reason that we don't recommend similarly starting with just the end reads and establishing those rather than tossing them into the whole mass-phrap thing? In the project I'm looking at (admittedly the worst-looking of the ones I picked, to hopefully head off student complaints because, hey, look, you might have pulled *this* project!) there are 56 end reads spread out over 6 of the 7 contigs. Phrapping the end reads separately seems like it ought to be helpful in a situation like this....
Chris Jones
Assoc. Prof. of Biology
Moravian College
Bethlehem PA

cshaffer
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Re: Starting out from fosmid ends

Post by cshaffer » Tue Feb 05, 2013 7:19 pm

The basic Phrap protocol is to take each read individually and attempt to build an assembly, it starts with the reads that match most closely to each other and builds from there.

There is no way I know of to tell Phrap the naming scheme for ends and ask it to do the assembly in the way you suggest (from the oustide it). You could do this manually: Tear out all the end reads, run mini-assembly on all end reads and join all the other reads to these assemblies but that would be very labor intensive, as you would never be able to use Phrap again because it would reassemble using its best-> worst order of assembly.

cjones
Posts: 99
Joined: Sun Feb 04, 2007 10:19 pm
Location: Moravian College, Bethlehem PA
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Re: Starting out from fosmid ends

Post by cjones » Tue Feb 05, 2013 10:05 pm

Just to see if it helped me orient, I pulled all 56 end reads from their contigs and tried to run miniassembly on them. I got an error message saying "phredPhrap was supposed to put the name of the new ace file in a file called mini.130205.165339.fof. However, this file cannot be opened. Thus we cannot merge this new assembly." I get the same thing every time, even if I choose only a subset of the end reads to mini-assemble (the second 6-digit string is different each time, but the date string remains the same). I don't see anything in the GEP Consed guides about reassembly problems; any suggestions?
Chris Jones
Assoc. Prof. of Biology
Moravian College
Bethlehem PA

cshaffer
Posts: 211
Joined: Sun Feb 04, 2007 10:29 pm
Location: Washington University in St Louis
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Re: Starting out from fosmid ends

Post by cshaffer » Wed Feb 06, 2013 3:33 am

can you go back to the terminal and copy/paste the error message consed prints out. The message there is usually longer and more informative.
thanks

cshaffer
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Re: Starting out from fosmid ends

Post by cshaffer » Wed Feb 06, 2013 5:11 pm

Oh another thought,

not all end reads in a project are reads from that fosmid. There should be at least two other sets of end reads that come from the ends of the adjacent clones. The name of the end read includes "fosmidend" and then has the name of the template and a code for who did the sequencing and of course ends in .b1 or .g1 depending on if its the forward or reverse primer. To try mini-assembly I would recommend doing one set at a time. so all the .b1 with the same template, then another with all the g1 and template etc.

chris

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