[boetsie]

Hi again,

I think i know what the problem is. You have a library called "/home/carroll/Desktop/data_carroll/SSPACEtests/leo95130_I". This is a very strange name for a library. Name it something like "leo95130_I" or "lib1" (without the quotes though). Now, with your current library name, the script will try to create a file containing this library name in folder 'reads'. It will now be something like;

reads/home/carroll/Desktop/data_carroll/SSPACEtests/leo95130_I.filtered.reads

This will surely cause problems (as you noticed). The other error you get is probably caused by the same problem, namely your library name.

Your library should be something like;

library1 /path-to-file/filename_1.fastq /path-to-file/filename_2.fastq 500 0.25 0

If you are unable to generate the library, you can mail me your current library file and i can help you.

Kind regards,
Boetsie

[themwg]

the next problem

Thanks Boetsie for the quick reply.
Sure enough I get further along if I just direct to SSPACE.pl from my directory. However I hit a second problem during the Reading, filtering and converting input seqs it Can't write to single file. here it is below

=>Fri Feb 11 11:55:38 2011: Reading, filtering and converting input sequences of library '/home/carroll/Desktop/data_carroll/SSPACEtests/leo95130_I' initiated
Can't write to single file -- fatal

=>Fri Feb 11 11:55:38 2011: Storing contigs to format for scaffolding

LIBRARY /home/carroll/Desktop/data_carroll/SSPACEtests/leo95130_I
------------------------------------------------------------

=>Fri Feb 11 11:55:44 2011: Building Bowtie index for contigs (tmp.standard_output/subset_contigs.fasta)

Bowtie-build error; -1 at /opt/SSPACE-1.1_linux-x86_64/bin/mapWithBowtie.pl line 37.
WARNING: No scaffolding, because no reads found on contigs

I imagine the bowtie build error is related to the first. Any thoughts on why it can't write to the single file (merging the two seq files?). Those files are in fastq format from illumina. They are also both quite large >10GB. My machine has a meager 44GB Ram. IF any of that is at all relevant here.

Thanks!

[boetsie]

Hi themwg,

good that it is working! Unfortunately, you can't specify where the folders end up. The folder structure is generated in your current working directory. Maybe you can turn the problem around; go to the directory where you would like the files/folders will end up and run the program from there. Then specify the full path to the contigs and also the full paths in the library file for your paired sequences.

If this won't work, i'm able to make a customised script for you You can mail me any time.

Boetsie

Hi, I'm excited to get SSPACE up and running. Unfortunately I'm getting a permission denial when making the directories (line 141). SSPACE is installed on a server in a directory where I don't have write permissions, which I suspect is the problem. Is there a way to direct where the results folders end up? or is my issue much simpler (and dumber).

[themwg]

Hi, I'm excited to get SSPACE up and running. Unfortunately I'm getting a permission denial when making the directories (line 141). SSPACE is installed on a server in a directory where I don't have write permissions, which I suspect is the problem. Is there a way to direct where the results folders end up? or is my issue much simpler (and dumber).

[e-summer-3]

Hi, boetsie.
SSPACE is very good tool for scaffolding. I thanks you for your good job.

By the way, How does SSAPCE pronounce? "espeis"?

[boetsie]

I am using SSPACE and I find this tool very useful and user friendly (not as Bambus!).

Thanks!

Thank you for this compliment

[gstitan]

congratulation

I am using SSPACE and I find this tool very useful and user friendly (not as Bambus!).

Thanks!

[boetsie]

Hi Corthay,

no problem, good that it is clear now

1)
Hmmm, that should never be the case. Are you looking at the summary file to conclude that the total bases of scaffolds is increased? Because this value (sum (bp)) is the total number of bases WITH N's. The number of bases without N's should either be the same or less than the original total number of bases, since it tries to merge the contigs if they share -n overlap.

If you want, i can send you a script which calculates the number of N's in the scaffolds.

2)
For estimating the gap, i use the size of gap using reads.

Kind regards and no problem for the questions ,
Boetsie

Hi boetsie,

Thanks for your quick reply. I understood how uniqueness is guaranteed.
Then, I have two more questions please.

Firstly, I am wondering why the total bases of scaffolds without N is increased even though I set 0 for "-x" option.

Secondly, how do you calculate the distance of reads within a given contig pair.
Do you estimate the size of gap using reads, or gap size is just ignored ?

Sorry for asking so many questions.

Thanks
Corthay.

[corthay]

Hi boetsie,

Thanks for your quick reply. I understood how uniqueness is guaranteed.
Then, I have two more questions please.

Firstly, I am wondering why the total bases of scaffolds without N is increased even though I set 0 for "-x" option.

Secondly, how do you calculate the distance of reads within a given contig pair.
Do you estimate the size of gap using reads, or gap size is just ignored ?

Sorry for asking so many questions.

Thanks
Corthay.

Hi again

I indeed use only reads that can uniquely map to only one position on all the contigs. I use the option -m 1 from Bowtie (see; http://bowtie-bio.sourceforge.net/ma...html#reporting). Otherwise, it is impossible to know what link should be made if a read maps to multiple contigs.

Is this what you mean?

Kind regards,
Boetsie

[boetsie]

Hi boetsie again,

I would like to ask you if only unique mapped reads are used for the scaffolding.

If not, I am planing to mask repeat sequence before scaffolding.

Thanks,
Corthay

Hi again

I indeed use only reads that can uniquely map to only one position on all the contigs. I use the option -m 1 from Bowtie (see; http://bowtie-bio.sourceforge.net/ma...html#reporting). Otherwise, it is impossible to know what link should be made if a read maps to multiple contigs.

Is this what you mean?

Kind regards,
Boetsie

[corthay]

Hi boetsie again,

I would like to ask you if only unique mapped reads are used for the scaffolding.

If not, I am planing to mask repeat sequence before scaffolding.

Thanks,
Corthay

[boetsie]

What you need to do is track the positions of these features from the input contigs onto the output scaffolds to internally generate a new tab-delimited input file with the right coordinates... I tried doing this with BioPerl, but unfortunately got tied in knots with the cryptic class hierarchy.

In theory it shouldn't be hard to say 'position x on contig y in the input is now position j on scaffold k in the output', and simply run it again for the new library. However, I guess there is quite a bit of complexity to such a code.


Anyway, just a suggestion for improvement of an already useful tool!

Cheers,
Dan.

Hi Dan,

first of all, thank you for the suggestions and the positive feedback!

I see what you mean, and i think it is indeed a useful function to allow other input formats. I think as a start it would be nice to allow .sam format inputs.

About remembering the positions i'm doing quite the same with remembering which contigs are on which scaffolds after each library. I think the same trick could be applied for mapping.
I'll see what i can do.

Thanks,
Boetsie

[dan]

... since we do an hierarchical clustering ... for each library we align the reads to the new scaffolds, therefore, no predefined paired sequence alignments could be provided ...

What you need to do is track the positions of these features from the input contigs onto the output scaffolds to internally generate a new tab-delimited input file with the right coordinates... I tried doing this with BioPerl, but unfortunately got tied in knots with the cryptic class hierarchy.

In theory it shouldn't be hard to say 'position x on contig y in the input is now position j on scaffold k in the output', and simply run it again for the new library. However, I guess there is quite a bit of complexity to such a code.


Anyway, just a suggestion for improvement of an already useful tool!

Cheers,
Dan.

[boetsie]

Hi Boetsie,

Thanks for the response.

I've just specified "k=2" as clone coverage of BAC ends is almost 5x.
As a result, scaffolds N50 is a bit improved and the number of scaffolds is reduced. Thanks for the development of useful tool.

Corthay.

Hi Corthay,

great that it worked and that it improved your assembly a bit!

Kind regards,
Boetsie

[boetsie]

What would be great is a simple tab delimited format for providing paired sequence alignments, rather than going via Bowtie... I had a quick look at the code, but unfortunately I couldn't work out where to add such functionality easily. I'll have another look at some point if nobody else does.

Hi Dan,

i know what you mean, but than multiple library input can't be used since we do an hierarchical clustering (first generate scaffolds using one library, than produce scaffolds by aligning next library on first scaffolds and produce new scaffolds etc...). So for each library we align the reads to the new scaffolds. Therefore, no predefined paired sequence alignments could be provided, except if only one library is used. In addition, if we have such an input we would be very similar to Bambus. Our purpose is to have an easy to use scaffolder without providing complex input formats, but with a simple fasta input.

Next week, i'll try to provide another alignment tool (e.g. Newbler) to map long reads to the contigs/scaffolds.

Kind regards,
Boetsie