HELP! with forward reverse constraints
Bee
I am trying to use forward and reverse constraints in the assembly
setup options. I want to assemble a bunch of sequences with their
reverse complements.
I have a bunch of sequences named xxx_F, xxx_R, yyy_F, yyy_R, etc. and
I want to assemble xxx_F with xxx_R and yyy_F with yyy_R
automatically. I have tried to do this by selecting "use forward and
reverse constraints" in the assembly setup box and setting my forward
indicator to F, reverse indicator to R, and the delimiter to _. I
thought that when I then go and assemble all of my molecules at once,
it will know that xxx_F and xxx_R should go together and yyy_F and
yyy_R should go together, etc. But it is not doing that. Am I
missing the point of this function? Is there any way to do what I'm
trying to do?
Invitrogen tech support won't help me because I don't have a service
contract, but someone out there must have tried to do the same thing,
right?
If anyone out there knows how to do this, I would be so so so
thankful.
Thanks
Bee
malc
assembly is incorrect; the constraint should prohibit contigs to be
considered that assemble a forward read with its corresponding reverse
read unless they are within the range specified and unless one of them
is reverse complemented.
Does this help?
>From Contig Express helpfile you can read:
Use Forward-Reverse Constraints: When reads from both ends of
subclones are available, constraints are satisfied if they lie on
opposite strands of a double-stranded DNA molecule and within a
specified minimum and maximum range. This corrects assembly errors due
to misplacement of reads containing repeat sequences and minimizes
occurrence of singletons. A few unmet constraints are allowed.
However, if a sufficient number of constraints are not satisfied by a
join AND they all suggest an alternative one supported by the overlap
information, the alternative join will be made. For most small- and
moderate-sized projects, it is not necessary to use this feature,
unless in a situation involving large region of repeats. Default: OFF.
Alos, for more documentation, it helps to know that Contig Express
employs the CAP3 alogorithm, whose documentation should help further.
>From http://genome.cs.mtu.edu/cap/cap3.html we read:
"A forward-reverse constraint is often produced by sequencing both
ends of a subclone. A forward-reverse constraint specifies that the
two reads should be on the opposite strands of the DNA molecule within
a specified range of distance. CAP3 makes use of a large number of
forward-reverse constraints to locate and correct errors in layout of
sequence reads. This capability allows CAP3 to address assembly errors
due to repeats. CAP3 also uses constraints to link contigs separated
by a gap. This feature provides useful information to sequence
finishers. The algorithm used in CAP3 is designed to tolerate wrong
constraints, which are due to errors in naming and lane tracking. "
HTH,
Malcolm
Bee
Thanks this does clarify a lot for me, and now I see what you mean. I
guess I am still wondering though, is there a way to "batch" create
contigs from F and R reads from samples with a similar name??? I know
you can do this in Sequencher and it would make my life VERY MUCH
easier.
But I guess maybe not in Vector NTI... it is funny, I got this message
from Invitrogen when I tried to email the exact same question to their
tech support (like I said, I don't have a support contract)..
"Hello,
I believe that you called yesterday asking the same question but I
told you we were unable to offer service because Yale did not have a
support contract with us. I took a deep look at your issue
nevertheless and it turned out to be that the software was actually at
fault not performing the the expected task at all. You didn't do
anything wrong. So, I owe you an apology and I thank you for bringing
up this issue to us. We must fix this as a software bug. The fix
should be available in our next release but can't give your a time-
table yet."
Weeeird....
On Apr 5, 3:56 pm, "malc" <malcolm.c...@gmail.com> wrote:
> I think your understanding of how the contraints are used to guide the
> assembly is incorrect; the constraint should prohibit contigs to be
> considered that assemble a forward read with its corresponding reverse
> read unless they are within the range specified and unless one of them
> is reverse complemented.
>
> Does this help?
>
> >From Contig Express helpfile you can read:
>
> Use Forward-Reverse Constraints: When reads from both ends of
> subclones are available, constraints are satisfied if they lie on
> opposite strands of a double-stranded DNA molecule and within a
> specified minimum and maximum range. This corrects assembly errors due
> to misplacement of reads containing repeat sequences and minimizes
> occurrence of singletons. A few unmet constraints are allowed.
> However, if a sufficient number of constraints are not satisfied by a
> join AND they all suggest an alternative one supported by the overlap
> information, the alternative join will be made. For most small- and
> moderate-sized projects, it is not necessary to use this feature,
> unless in a situation involving large region of repeats. Default: OFF.
>
> Alos, for more documentation, it helps to know that Contig Express
> employs the CAP3 alogorithm, whose documentation should help further.
>
> >Fromhttp://genome.cs.mtu.edu/cap/cap3.htmlwe read:
Cook, Malcolm
which lists all the constraints which allows you to get around using any
particular naming convention. See
http://genome.cs.mtu.edu/cap/cap3.html
But, Contig Express seems not to allow you to set this parameter.
I do not understand the reply you got from support.
Good luck.
Malcolm Cook