[Automated-sequencing] Re: Need help using phred , cross_match and phrap

Phillip San Miguel via autoseq%40net.bio.net (by pmiguel At purdue.edu)
Thu Nov 9 15:18:30 EST 2006

talanki wrote:
> Hi,
> I am trying to assemble sequences using the phred,cross_match and phrap
> softwares. I read the manuals and am a little bit confused on how to
> use them.
> I have 2 sequence files
> 1.  abc_F.abi  - forward read i.e read from 5' to 3' on the sense
> strand and the other is,
> 2.  abc_R.abi  - reverse read i.e read from 5' to 3' on the antisense
> strand.
> I need to create a contig of these two sequences.
> These are the following steps I performed.
> 1)
> I used phred and created the following files
> abc_F.abi.seq
> abc_F.abi.qual
> abc_R.abi.seq
> abc_R.abi.qual


> My first doubt is how to assemble the above 2 sequences using phrap.
> Should I write both the sequences in tht single file?

Yes. Also the quals should be in a single file. Actually phred can do 
this for you with the -sa and -qa options. Eg:

phred -if my_input_traces_filelist.txt -sa abc.fasta -qa abc.fasta.qual

> What should be the naming convention of the file?

phrap doesn't care. But if your .qual file has the exact same name as 
the sequence + a ".qual" suffix, then phrap will automatically use the 
quality values to help it assemble. Otherwise the quality values will 
not be used.

> Can I get a brief description on how to use phrap. the phrap
> documentation is confusing and I am not able to understand.

phrap abc.fasta

phrap attempts to assemble all the sequences in "abc.fasta" using the 
quality values in "abc.fasta.qual" if such a file exists. Among the 
output files created will be a file of contig sequences and a file of 
singlet (unassembled) sequences.

> Can anybody help me?

No need to do the vector cross_matching for just F/R reads from a single 
  clone. If you do want cross_match to help you identify vector, I would 
suggest you run cross_match on the contig after assembly.

phrap is pretty high-powered--typically one uses it to assemble hundreds 
or thousands of reads. But I do use it for simple F/R read assemblies.

www.phrap.org has detailed documentation for phred, phrap and consed. 
But much of it requires fairly in-depth study to use if you are not 
already familiar with the programs.

> bye,
> LT

Feel free to ask more questions if this doesn't work for you.

Phillip SanMiguel
Purdue Genomics Core Facility

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