[boetsie]

Hi again Ashu,

1. yes, this is possible. I've used both assemblies for scaffolding before, including paired-end and/or matepair reads.

2. Could you maybe send me your libraryfile and summaryfile as private message, so i can check what is going on.

Regards,
Boetsie

[Ashu]

Hi Boetsie,
I am trying to use SSPACE, for scaffolding with out any success, I have the following questions for you:
1 Does it use contigs from ABySS assemblyor CLCbio, and add paired end and mate pair??
2 Why am I getting empty result, with no improvement in N50 or anything. I have tested every possible option, -k2 -k5
can you please give some suggestions about it.

Thank you,
with kind regards,
Ashu

[boetsie]

Hi Boetsie,

Just out of curiosity is this script written in 32 bit perl? I see an issue with the program not taking beyond a certain level of memory.

Yes, i think it is written in 32 bit..

[boetsie]

Hi

I have a problem of using SSPACE. the process was failed at bowtie-build. I can use bowtie to do index with my genome file, but it seems not working in SSPACE... What's the problem?

Thanks

I quote an older post in this thread;

The problem was mainly solved by going through the directory were the main SSPACE script (SSPACE_v1-x.pl) and folders are stored using the command line. Then, do one of the following;

chmod a+x bowtie/*

or

chmod 777 *

in your command line.

If this won't work, then you may try to download the newest Bowtie version at http://sourceforge.net/projects/bowt...bowtie/0.12.7/

Replace the files in the bowtie folder with the ones you've downloaded.

Kind regards,
Boetsie

[christinawu2008]

problem with SSPACE

Hi

I have a problem of using SSPACE. the process was failed at bowtie-build. I can use bowtie to do index with my genome file, but it seems not working in SSPACE... What's the problem?

Thanks

[KanyeDidIt]

Hi Boetsie,

Just out of curiosity is this script written in 32 bit perl? I see an issue with the program not taking beyond a certain level of memory.

[boetsie]

Thank you for the suggestion Vidja, appreciate your help. However, right now i think including an independent script for conversion of .sam to .tab would be the best idea. SAM input can be possible, but I have to find a way to find the paired reads. With bowtie I do this by including /1 and /2 to the reads, so I know which is the left and right read (hence; i do a single read mapping, because Bowtie only finds read-pairs on same contig). Readnames differ for each platform, so I should probably let the user include the postfix of both reads. That's why i think at the moment .tab is the best way.

Conclusion; have to think about it

[VidJa]

Hi Boetsie,

great explanation, I expected sam/bam to be slower. Another nice aligner which could be considered is PASS: http://pass.cribi.unipd.it/cgi-bin/pass.pl
Very fast and has its own output type or gff, besides sam/bam. It has an option to attempt contig association and handles long reads.

[jstjohn]

Nice tests! Good to see how that /dev/fd stuff works.

[westerman]

Actually on most *nix systems including mac and every linux I have worked on so far you can use "/dev/fd/0" as the file name when you want to output something to standard out and the program doesn't give that option.

For example:
echo "hello world">/dev/fd/0

Shouldn't that be /dev/fd/1 ?? file descriptor #0 is usually stdin. Granted /0 works but I think that this is a side effect and not something to be relied on.

Besides that example proves nothing since you are taking stdout and (if you use /dev/fd/1) putting it into stdout. What you need for proof is a program like:

#!/usr/bin/perl
open (TEST, '>', $ARGV[0]) or die "Can not open file $ARGV[0]\n";
print TEST "This is a test\n";
exit;

If I name the above 't.pl' then I can run the following 4 examples:

1) if there is no file name given then nothing is output; either in a file nor in stdout.

rm -f test.tmp; ls test.tmp; ./t.pl ; ls test.tmp
ls: test.tmp: No such file or directory
ls: test.tmp: No such file or directory

2) If a file name is given then the file is created but no stdout.

rm -f test.tmp; ls test.tmp; ./t.pl test.tmp ; ls test.tmp
ls: test.tmp: No such file or directory
test.tmp

3) If I give the stdout file descriptor as the file name then I get text on stdout but not in the file.

 rm -f test.tmp; ls test.tmp; ./t.pl /dev/fd/1 ; ls test.tmp
ls: test.tmp: No such file or directory
This is a test
ls: test.tmp: No such file or directory

4) Likewise I can use the stdin file descriptor

rm -f test.tmp; ls test.tmp; ./t.pl /dev/fd/0 ; ls test.tmp
ls: test.tmp: No such file or directory
This is a test
ls: test.tmp: No such file or directory

5) But the above to descriptor #0 is really a side effect as can be shown by piping stdout to another program. In this case I'll use 'od' (octal dump). Using descriptor #1 (stdout -- the recommended descriptor) I get 'od' output.

 rm -f test.tmp; ls test.tmp; ./t.pl /dev/fd/1 | od -c; ls test.tmp
ls: test.tmp: No such file or directory
0000000   T   h   i   s       i   s       a       t   e   s   t  \n
0000017
ls: test.tmp: No such file or directory

6) Whereas if I use the incorrect descriptor #0 (stdin) I do not get proper output but instead just see the text.

 rm -f test.tmp; ls test.tmp; ./t.pl /dev/fd/0 | od -c; ls test.tmp
ls: test.tmp: No such file or directory
This is a test
0000000
ls: test.tmp: No such file or directory

Whew! Now back to what I was doing. Which is running sspace in various configurations.

[jstjohn]

BWA for example can output SAM to stdout if you don't give it the -o outfile.sam option.
-John

Actually on most *nix systems including mac and every linux I have worked on so far you can use "/dev/fd/0" as the file name when you want to output something to standard out and the program doesn't give that option.

For example:
echo "hello world">/dev/fd/0

[jstjohn]

SAM output will generate output for all reads, no matter they are mapped against a contig or not. Bowtie output only contains reads that are mapped. Since SSPACE only contains a fraction of the contigs (only begin and ends of each contig), the number of reads that maps is also low. SSPACE goes through all output lines, thus the higher the number of output lines, the slower the program goes.
Boetsie

Hi Boetsie,
I don't know about all mappers that output SAM format, but BWA for example can output SAM to stdout if you don't give it the -o outfile.sam option. If you are only interested in reads that map, or reads where only one mate of the pair maps, you can either pipe the output through samtools and filter on the flag, or pipe through a perl or awk one liner that filters the sam output on the flag. The flag contains info about whether the read maps and/or the mate maps.
-John

[boetsie]

Hi Boetie,

Do you plan to include the use of SAM/BAM as output instead of the default bowtie, that way you clould dropin all sam/bam capable aligners, like BWA (for longer reads with the sw option), Smalt http://www.sanger.ac.uk/resources/software/smalt/
or Stampy http://www.well.ox.ac.uk/project-stampy
and probably a full bag of other aligners.

After some testing with Bowtie, i came to the conclusion that going for a SAM/BAM output instead of the default bowtie output is much slower.

SAM output will generate output for all reads, no matter they are mapped against a contig or not. Bowtie output only contains reads that are mapped. Since SSPACE only contains a fraction of the contigs (only begin and ends of each contig), the number of reads that maps is also low. SSPACE goes through all output lines, thus the higher the number of output lines, the slower the program goes.

Say there are 1M reads, and only 10.000 reads map. Bowtie will generate 10.000 output lines, while SAM produces 1M output lines, making it 100 times slower to read in the output file.

Therefore, I'll make it possible to insert tab-delimited files with information about paired reads in the format;

<read1_tig> <read1_start> <read1_end> <read2_tig> <read2_start> <read2_end>

I will provide a script that can convert SAM output files to a TAB file. This way, all SAM capable aligners can be used.

In addition, multiple TAB files of different libraries can be given, as well as a combination of TAB and normal paired-reads. For example; if you have a paired-end library of 200bp and one with 500bp. For both libraries you map the reads to the contigs, generating two SAM files, which you can convert to .tab file. Both could be given to SSPACE, first SSPACE scaffolds the contigs using the 200bp library. Next, the positions of the contigs are updated by determining their new position within the scaffolds. Then, the 500bp library is used for scaffolding the previous scaffolds generated with the 200bp library.

Still in testing fase though, but the results till now look ok. I get similar results if i input a paired-end fastQ file, or a .tab file.

I'll keep you updated!

Kind regards,
Boetsie

[KanyeDidIt]

edited after solution

[boetsie]

Hi Vidja,

yes, i'm currently working on this though i don't have much time. Thank you for the suggestion of BWA for longer reads! I hope I can include more aligners.

Boetsie

Hi Boetie,

Do you plan to include the use of SAM/BAM as output instead of the default bowtie, that way you clould dropin all sam/bam capable aligners, like BWA (for longer reads with the sw option), Smalt http://www.sanger.ac.uk/resources/software/smalt/
or Stampy http://www.well.ox.ac.uk/project-stampy
and probably a full bag of other aligners.