CAP3 parameters

[BOYD]

Hello, I'm trying to use cap3 to assemble a number of long (>1kb) sequences with short primer
sequences that are typically 16-30 bp in length. I have tried many variations of the cap3
parameters and I either get the primers assembling together into contigs, or the longer
sequences assembling. Is there any way to relax the stringency of the cap3 parameters,
so that everything assembles?

If cap3 is not capable of doing this, are there other command line assemblers that can
assemble from a multiple FASTA (without associated .qual files)??

[Eric Fournier]

You could use a program like SEQClean to remove the primers from your sequences. This should increase the quality of your assembly.

[BOYD]

I am afraid you misunderstood the question - I should probably have been more explicit.
I have multiple 500bp - 1kb sequences AND short 15-30bp Primer sequences in FASTA format which I would like to assemble together into a contig, so I can see where my
primers are located within the contig.............

[Eric Fournier]

Your question does not make sense.

If you want to know where your primers would fit on your sequences, then you need to BLAT/BLAST/Some-Alignment-Tool them. This is not an assembly problem, and CAP3 is not the tool for the job.

[BOYD]

I want to generate an .ace assembly file or alignment file, so I can
see where my primers align with respect to the longer dna sequences.
There will be snps present and I wanted to be able to examine where
the primers are located compared to snp positions (and hopefully parse
this data). I find it hard to believe that there isn't a command line assembler
that can do this, after all I can assemble long reads and primer sequences
in Sequencher and DNAstar. There must be a way of relaxing parameters
sufficiently in cap3 or phrap to allow assembly of 1kb+ sequences and
short 14-30bp sequences.

[Eric Fournier]

I find it hard to believe that there isn't a command line assembler
that can do this,

What you want to do is not what assemblers are meant to do, so it's not surprising that none of them behave as you expect them to. Assemblers are meant to assemble genomes/transcriptomes out of similarily sized reads. Aligning primers to long reads is not part of their feature list.

What you should do is assemble your long reads (preferably with an assembler that detects SNPs, like MIRA) and then align your primers with another software like BLAST.

[DZhang]

Hi,

you may try DNAStar. It should work for you but it is a commercial software. (I am not affiliated with it).

Douglas

https://www.contigexpress.com

[BOYD]

Finally managed to do what I wanted using Phil Green's phrap assembler, and
relaxing the parameters, which were:

-default_qual 1
-forcelevel 10
-minmatch 14
-minscore 10
-force_high

Thanks for everybody's suggestions.