Velvet Question

[VNou]

Hello, i am a new Velvet user and i am trying to assemble some libraries that i found in this site:

http://gage.cbcb.umd.edu/index.html

I have some question about Velvet:

1) Shuffle operation is needed always when more than 1 library is assembled? If is not needed always in what it helps?

2)I saw this operation in the recipes of the site i mentioned:
shuffleSequences_fastq.pl shortjump_1.fastq shortjump_2.fastq - | fastx_reverse_complement > shortjump.fastq
What - | fastx_reverse_complement > shortjump.fastq is needed for?

3)Using the the command above i have the following error:
/var/spool/slurmd/job414800/slurm_script: line 21: fastx_reverse_complement: command not found
Why this error is produced? Is it a problem of slurm? Can i fix it or there is another alternative?

4) I also tried to use:
fastq -shortPaired frag.fastq -shortPaired2 shortjump.fastq -shortPaired3 longjump.fastq
but i have a problem with -shortPaired3. I have to put at max 2 parameters?

Thank you a lot

[rahularjun86]

Hi,
1). Shuffle script is to convert two paired files to single file readable by velveth. Please go through this link: http://www.molecularevolution.org/re...owtie_activity
2). I have not tried this before, you can refer the fastx tools(http://hannonlab.cshl.edu/fastx_toolkit/), and see the command used, it is for Producing the Reverse-complement of each sequence in a FASTQ/FASTA file.
3). "fastx_reverse_complement: command not found" because you do not have fastx installed on your system. But why do you want to Reverse-complement your sequences ???
4). Your velvet installation is not supporting the 3rd library, Please re-install velvet with >2-3 libraries. And refer the section "2.3.2 CATEGORIES" of velevt manual "http://helix.nih.gov/Applications/velvet_manual.pdf". By default, there are only two short read categories, but this variable can be extended to your needs.
Must go through this link: http://www.molecularevolution.org/re...owtie_activity.
Best wishes,
Rahul

[VNou]

First of all thanks a lot for your answers.
I want to ask something more about the command :
- | fastx_reverse_complement > shortjump.fastq. Instead of installing fastx is there any other way to do it? Why here http://gage.cbcb.umd.edu/recipes/velvet.html the use it?
I assembled without using it and just shuffling the 2 short jump libraries but i get in the case of Rhodobacter sphaeroides a N50 of 27bp and i think is too small. The input parameters i used are the same with the site(k-mer 31 exp_cov auto). They obtain i much bigger N50 358kbp. I think the problem is related to the reverse complement. Do you have any idea about what can i do to solve the problem without install fastx tools because i encounter difficulties in installing them?
Thanks again a lot