[mattoslmp]

I have a doubt

Dear Boetsie, please I would like known if with SSPACE, can I use contigs + pair-end reads for extension of an assembled. I have obtained a set of contigs of one genome of NCBI, and I would like to extend of theses contigs using pair-end reads obtained of illumina, because this assembled of NCBI is only parcial.

Thanks for any help anf by your Attention


Mattos LP.

Hi all,

during my Master thesis I developed a stand-alone scaffolding tool named SSPACE for scaffolding pre-assembled contigs using paired-read data. I developed this program since I couldn't find a program which was able to do this, except from Bambus. However, we had lots of issues on Bambus, including errors and complicated input datasets.

Therefore, SSPACE was developed. The main featues are;

* Inputs are simple FASTA contig sequences as well as (multiple) FASTA/FASTQ paired-read data
* High-quality scaffolds in a short runtime and limited memory requirements
* High reduction of the amount of contigs stored into scaffolds and high N50 value
* Multiple library input of both paired-end and/or mate pair datasets
* Possible contig extension of unmapped sequence reads
* Easy interpretation of the final scaffolds
* Visualization of the final scaffolds using GraphViz

SSPACE has been tested on the E.coli, Grosmannia clavigera and Giant Panda genomes and showed less scaffolds and higher N50 value compared with the produced scaffolds from common de novo assemblers, like Abyss and SOAPdeNovo.

SSPACE is freely available at

http://www.baseclear.com/sequencing/data-analysis/bioinformatics-tools/sspace/

The publication is accepted at bioinformatics and will be online soon. Publication shows more detailed information about the produced scaffolds and their quality, including time and memory information.

Hope it could be useful and any comments or questions are ofcourse welcome.

Cheers,
Boetsie

[boetsie]

Hi Mattos LP,

This should not be a problem, as long as the input (contig) sequences are in fasta format. SSPACE tries to extend the end of the sequences using the unmapped sequence reads.

Boetsie

[jpummil]

Yup, data info below:
Current Filename File Size Encoding Length Insert Size Type
L001_R1_MiSeq.fastq 16GB Illumina 1.9 300bp 479 Pair Ended
L001_R2_MiSeq.fastq 16GB Illumina 1.9 300bp 479 Pair Ended

L003_R1_MP.fastq 21GB Illumina 1.9 101bp 6000 Mate Pairs
L003_R2_MP.fastq 21GB Illumina 1.9 101bp 6000 Mate Pairs

s1_R1_PE.fastq 27GB Illumina 1.5 101bp 150 Pair Ended
s1_R2_PE.fastq 27GB Illumina 1.5 101bp 150 Pair Ended

s2_R1_PE.fastq 24GB Illumina 1.5 101bp 150 Pair Ended
s2_R2_PE.fastq 24GB Illumina 1.5 101bp 150 Pair Ended


I'll try a couple with BWA and see what happens.

So, either I am doing something incorrectly in my parameter specifications for SSPACE, or there is an issue with my assembly.

Switching to BWA gave the same error: Process 'extend/format contigs' failed on Sat Dec 20 17:43:43 2014

In this attempt, I was using the Mate Pair libs alone to try and improve the assembly...but in previous attempts, I have used the Pair Ended libs. Incidentally, these libraries are all included in the original assembly (with Velvet and with Ray).

[jpummil@razor-l3 Snake_v2]$ more libraries_alt.txt
lib1 bwa /scratch/jpummil/Snake_v2/L003_R1_MP-RC.fastq /scratch/jpummil/Snake_v2/L003_R2_MP-RC.fastq 6000 0.50 FR

Command line:
perl /home/jpummil/SSPACE-STANDARD-3.0_linux-x86_64/SSPACE_Standard_v3.0.pl -l libraries.txt -s Ray-53-contigs.fasta -x 1 -T 32 -b Ray_standard_out_v2_extend_bwa

After a ~20hr run time using 32 cores and 301GB of ram...

[jpummil@razor-l3 Snake_v2]$ more SSPACE.980772.sched
Your inserted inputs on [SSPACE_Standard_v3.0_linux] at Fri Dec 19 22:08:29 2014:
Required inputs:
-l = libraries.txt
Number of paired files = 3
-s = Ray-53-contigs.fasta
-b = Ray_standard_out_v2_extend_bwa

Optional inputs:
-x = 1
-z = 0
-k = 5
-g = 0
-a = 0.7
-n = 15
-T = 32
-p = 0

Contig extension inputs:
-o = 20
-m = 32
-r = 0.9


=>Fri Dec 19 22:08:29 2014: Reading, filtering and converting input sequences of library file initiated
Reading read-pairs lib3.1 @ 6000000
Reading read-pairs lib2.1 @ 1300000
Reading read-pairs lib2.1 @ 20000000
Reading read-pairs lib3.1 @ 28000000
Reading read-pairs lib1.1 @ 21000000
Reading read-pairs lib1.1 @ 25000000
Reading read-pairs lib2.1 @ 49000000
Reading read-pairs lib2.1 @ 58000000
Reading read-pairs lib2.1 @ 67000000
Reading read-pairs lib2.1 @ 76000000
Reading read-pairs lib2.1 @ 86000000

------------------------------------------------------------

=>Fri Dec 19 22:23:15 2014: Extending of contigs with unmapped reads

=>Fri Dec 19 22:23:15 2014: Building BWA index for contigs
[bwt_gen] Finished constructing BWT in 281 iterations.

=>Fri Dec 19 22:48:22 2014: Mapping reads to contigs with BWA
**************************************************

Process 'extend/format contigs' failed on Sat Dec 20 17:43:43 2014


Anything obvious to anyone?

[fahmida]

Hi Boetsie,

As jpummil mentioned above I am also encountering similar error with SSPACE Standard v3.0.
I ran the following command with BOWTIE and BWA:

/software/SSPACE-STANDARD-3.0_linux-x86_64/SSPACE_Standard_v3.0.pl -l libraries.txt -s final.contigs.fasta -x 1 -T 8 -v 1

with BOWTIE I get the error:

=>Mon Feb 23 16:04:57 2015: Extending of contigs with unmapped reads

=>Mon Feb 23 16:04:57 2015: Building Bowtie index for contigs

=>Mon Feb 23 16:20:51 2015: Mapping reads to contigs with Bowtie
Thread 10 terminated abnormally: Can't open bwa output -- fatal
Thread 11 terminated abnormally: Can't open bwa output -- fatal
Thread 12 terminated abnormally: Can't open bwa output -- fatal
**************************************************
Process 'extend/format contigs' failed on Mon Feb 23 19:20:41 2015

with BWA I get the error:

------------------------------------------------------------

=>Tue Feb 24 10:31:47 2015: Extending of contigs with unmapped reads

=>Tue Feb 24 10:31:47 2015: Building BWA index for contigs
[bwt_gen] Finished constructing BWT in 159 iterations.

=>Tue Feb 24 10:40:54 2015: Mapping reads to contigs with BWA
**************************************************
Process 'extend/format contigs' failed on Tue Feb 24 13:06:41 2015

Any possible solution?

Best Regards.

[Votinhkiem90]

Hi all,
I have a contigs fasta file (denovo using velvet 1.2.0) and two set mate pair fastq files (5kb and 8kb). Now I want to make scaffold using contigs file and two set mate pair files, but I don't know any tools can do it.
Many thanks for suggest.

[Votinhkiem90]

Hi all,
I have a contigs fasta file (denovo using velvet 1.2.0) and two set mate pair fastq files (5kb and 8kb). Now I want to make scaffold using contigs file and two set mate pair files, but I don't know any tools can do it.
Many thanks for suggest.
p/s: Sequencer: illumnia

[ClemBuntu]

Hi Nathan, just came across the same issue, could you find a way to fix it?

Cheers

I also have the same problem as you guys.

Here the output I got with extension and using BWA :

Process 'extend/format contigs' failed on Wed Mar 4 20:02:03 2015

Of course no scaffold are generated and the "tmpbwa_logfile_extension" file which is in the tmp directory contains this ;

[main] CMD: /home/clement/Softs/SSPACE-STANDARD-3.0_linux-x86_64/bwa/bwa aln sspaceTry/alignoutput/sspaceTry.bwaIndexExt sspaceTry/reads/sspaceTry.Lib1.bwa.file1.26.fa
[main] Real time: 627.392 sec; CPU: 549.368 sec
Thread 19 terminated abnormally: Can't open bwa output -- fatal
Thread 21 terminated abnormally:
Bwa error; 0 at /home/clement/Softs/SSPACE-STANDARD-3.0_linux-x86_64/bin/ExtendOrFormatContigs.pl line 288.
Thread 22 terminated abnormally:
Bwa error; 0 at /home/clement/Softs/SSPACE-STANDARD-3.0_linux-x86_64/bin/ExtendOrFormatContigs.pl line 288.
Thread 23 terminated abnormally:
Bwa error; 0 at /home/clement/Softs/SSPACE-STANDARD-3.0_linux-x86_64/bin/ExtendOrFormatContigs.pl line 288.

could someone help me please ?

[fahmida]

I also have the same problem as you guys.

Here the output I got with extension and using BWA :


Of course no scaffold are generated and the "tmpbwa_logfile_extension" file which is in the tmp directory contains this ;




could someone help me please ?

Hi,

For the above bwa and bowtie issues, it seems to work fine with the default contig extension parameter i.e. -x 0.
The problem occurs if I want to extend the contigs during scaffolding i.e. -x 1.
Thanks Marten Boetzer for providing the solution.
(Through personal communication)

Regards.

[ClemBuntu]

Hi,

For the above bwa and bowtie issues, it seems to work fine with the default contig extension parameter i.e. -x 0.
The problem occurs if I want to extend the contigs during scaffolding i.e. -x 1.
Thanks Marten Boetzer for providing the solution.
(Through personal communication)

Regards.

Hi,

Indeed, that works with the -x 0 option. So maybe it's a silly question but why this option is made for ? Oo

Thanks

[fahmida]

Hi,

Indeed, that works with the -x 0 option. So maybe it's a silly question but why this option is made for ? Oo

Thanks

-x 1 worked fine with previous releases of SSPACE (from my own experience).
I am sure Marten is investigating the issue with the current version.

[illinu]

Same as few posts above, I am trying the new SSPACE_Standard_v3.0_linux and get the same problem, first:

Process 'extend/format contigs' failed

and when I do -x 0 I get this other error:

Process 'mapping reads' failed

Is this due to the version, I got it working fine a few months ago...

Can someone clarify what's going on?

[jpummil]

Same as few posts above, I am trying the new SSPACE_Standard_v3.0_linux and get the same problem, first:


and when I do -x 0 I get this other error:


Is this due to the version, I got it working fine a few months ago...

Can someone clarify what's going on?

I've never resolved this issue either. Really need to use the tool, but haven't been able to circumvent the error issue with -x 1

[ramesh.8v]

Can't locate getopts.pl

Hi everyone,
I'm running into "Can't locate getopts.pl" issue after my new perl installation. Couldn't able to solve this issue myself. I appreciate any help.

Thanks
Ramesh

[AdrianP]

Hello, see if this helps:

sudo cpan
install Perl4::CoreLibs

[ramesh.8v]

Thank you!
I have tried, but still running into the same problem.