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Old 07-28-2014, 02:03 PM   #201
fireant
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Quote:
Originally Posted by boetsie View Post
Hmmm, It seems that in the newest version of perl they removed the getopts.pl library (see http://search.cpan.org/~rjbs/perl-5....s_and_Pragmata). At this site they explain how to solve this issue:
http://heasarc.gsfc.nasa.gov/lheasoft/bugs.html

Hope this helps.
Boetsie
Anyone have an idea on this?
I looked at the nasa site and I am confused about how to solve the getopts.pl problem.
I found this on anothe page at the nasa site. The link to the fix it script is broken though....


Perl scripts with newer Perl:

"Many of the older Perl scripts in HEASoft (e.g. fhelp) require the getopt.pl or getopts.pl libraries which are no longer available in the newest Perl distributions, e.g. Perl 5.16.2 which ships with Fedora 18. This can result in the error "Can't locate getopt.pl in @INC..." when trying to run a script. To get around this, users may either download and install the Perl4::CoreLibs module from CPAN which includes getopt.pl, or download and run (in $HEADAS/../ftools/<architecture>/bin) a script which repairs our older Perl scripts to use Getopt::Std instead."



Nathan
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Old 08-06-2014, 07:24 AM   #202
lkral
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Last year i was able to run SSPACE just fine. Now I'm getting the "Can't locate getopt.pl in @INC…" error. Has anyone had success on a workaround? I'm on Mac OS X.

Thanks, Leos
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Old 08-06-2014, 08:03 AM   #203
maubp
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Nathan's post just above outlines two ways to fix this, the simpler one sounded like installing Perl4::CoreLibs module from CPAN.
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Old 08-06-2014, 12:43 PM   #204
lkral
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Quote:
Originally Posted by maubp View Post
Nathan's post just above outlines two ways to fix this, the simpler one sounded like installing Perl4::CoreLibs module from CPAN.
Thanks. I was hoping someone could verify that this worked before I started mucking with the system. Anyway, I did the install of the Perl4::CoreLibs module from CPAN and that fixed it.
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Old 08-07-2014, 07:33 AM   #205
fireant
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I now have another problem with the ExtendOrFormatContigs.pl script. Attempts to use bowtie failed due to problems with the output of the aligner and so I tried bwa as the aligner. However now after alignement I get this error call from the ExtendOrFormatContigs.pl script.
Bwa error; 0 at ExtendOrFormatContigs.pl line 288.

this is the line
system("$aligninput") == 0 || die "\nBwa error; $?" if($aligninput ne "");

Not sure what this means. My alignmentment folder looks fine.
I'm trying again without the extend contigs option but I sure would like to extend my contigs before scaffolding...

Nathan

Last edited by fireant; 08-07-2014 at 10:56 AM.
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Old 09-17-2014, 02:55 PM   #206
Mizzou55
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Hi,

Has anyone seen this problem, and know how to fix it. We were going to remove the offending reads, but the we don't know how to convert the read names in the Reads dir, back to the input read names for removal. Or is there something else we can do? These are BAC end sequences - 3730 reads.

Thanks,

Pat

Error: Read (read1ad27Error: Read (Error: Read (rError: Read (read891/1)
is less than 4 characters long
203/Error: Read (read74read815/2) is less than 3 characters long
2) is less than 4 characters long
8/2) is less than 3 characters long
ead1170/2) is less than 3 characters long
73/2) is less than 3 characters long07/2
) is less than 3 characters long
Error: Read (read1576/2) is less than 3 characters long
terminate called recursively
terminate called recursively
Error: Read (Error: Read (ead273/2) is less than 4 characters long
Error: Read (eadError: Read (read748/2) is less than 3 characters long
read207/2) is less than 3 characters long
295/2) is less than 3 characters long
Error: Read (read815/2) is less than 3 characters long
Error: Read (read891/1) is less than 4 characters long
Error: Read (read1073/2) is less than 3 characters long
Error: Read (read1170/2) is less than 3 characters long
terminate called after throwing an instance of 'terminate called
recursively
int'
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Old 12-16-2014, 02:55 PM   #207
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Quote:
Originally Posted by fireant View Post
Bwa error; 0 at ExtendOrFormatContigs.pl line 288.

this is the line
system("$aligninput") == 0 || die "\nBwa error; $?" if($aligninput ne "");

Not sure what this means. My alignmentment folder looks fine.
I'm trying again without the extend contigs option but I sure would like to extend my contigs before scaffolding...

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

Cheers
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Old 12-17-2014, 09:34 AM   #208
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Greetings all! Another plea for wisdom from SSPACE users...

So, I am working on a 1.7Gb diploid de novo genome assembly (http://seqanswers.com/forums/showthread.php?t=42555) and wanted to try and employ some enhanced scaffolding to improve continuity of some of my existing draft assemblies. However, I get an error message upon completion of the sspace run:
"Process 'extend/format contigs' failed on Sat Dec 13 07:49:06 2014"

I have accepted default values to start with on the majority of the settings:
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

Though sizeable (400GB memory and 18 hour runtime on 32 compute cores), not hitting any memory barriers, etc...job does complete and exit properly.

Think my libraries.txt is OK...
[jpummil@razor-l3 Snake_v2]$ more libraries.txt
lib1 bowtie /scratch/jpummil/Snake_v2/L001_R1_MiSeq.fastq /scratch/jpummil/Snake_v2/L001_R2_MiSeq.fastq 479 0.25 FR
lib2 bowtie /scratch/jpummil/Snake_v2/s1_R1_PE_Phred33.fastq /scratch/jpummil/Snake_v2/s1_R2_PE_Phred33.fastq 150 0.25 FR
lib3 bowtie /scratch/jpummil/Snake_v2/s2_R1_PE_Phred33.fastq /scratch/jpummil/Snake_v2/s2_R2_PE_Phred33.fastq 150 0.25 FR

Anything obvious come to mind? I had understood that with -x 1 flag, the assembly would be physically modified during the scaffolding process?

Thanks for any tips, and HAPPY HOLIDAYS!
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Old 12-17-2014, 10:40 PM   #209
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Quote:
Originally Posted by jpummil View Post
Think my libraries.txt is OK...
[jpummil@razor-l3 Snake_v2]$ more libraries.txt
lib1 bowtie /scratch/jpummil/Snake_v2/L001_R1_MiSeq.fastq /scratch/jpummil/Snake_v2/L001_R2_MiSeq.fastq 479 0.25 FR
lib2 bowtie /scratch/jpummil/Snake_v2/s1_R1_PE_Phred33.fastq /scratch/jpummil/Snake_v2/s1_R2_PE_Phred33.fastq 150 0.25 FR
lib3 bowtie /scratch/jpummil/Snake_v2/s2_R1_PE_Phred33.fastq /scratch/jpummil/Snake_v2/s2_R2_PE_Phred33.fastq 150 0.25 FR
Is your insert size really just 150 38 bp? 150 bp sounds more like your read length. As far as I recall, I never got SSPACE working with bowtie. However, it worked just fine with BWA..

So a library file would have been something like:

Code:
Org1 bwa Truseq_1_LRTrimmed.fastq Truseq_2_LRTrimmed.fastq 430 0.50 FR
These were 100 bp reads with 430 bp median fragment size..
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Last edited by rhinoceros; 12-17-2014 at 10:46 PM.
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Old 12-18-2014, 06:36 AM   #210
jpummil
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Quote:
Originally Posted by rhinoceros View Post
Is your insert size really just 150 38 bp? 150 bp sounds more like your read length. As far as I recall, I never got SSPACE working with bowtie. However, it worked just fine with BWA..

So a library file would have been something like:

Code:
Org1 bwa Truseq_1_LRTrimmed.fastq Truseq_2_LRTrimmed.fastq 430 0.50 FR
These were 100 bp reads with 430 bp median fragment size..
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.
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Old 12-29-2014, 07:58 PM   #211
mattoslmp
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Default 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.
Quote:
Originally Posted by boetsie View Post
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/...-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
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Old 12-31-2014, 01:47 AM   #212
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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
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Old 01-27-2015, 05:29 AM   #213
jpummil
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Quote:
Originally Posted by jpummil View Post
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?
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Old 02-23-2015, 05:37 PM   #214
fahmida
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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:
HTML Code:
/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:
HTML Code:
=>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:
HTML Code:
------------------------------------------------------------

=>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.
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Old 03-01-2015, 09:37 PM   #215
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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.
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Old 03-01-2015, 09:38 PM   #216
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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
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Old 03-04-2015, 10:58 PM   #217
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Quote:
Originally Posted by luiscunhamx View Post
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 :
Quote:
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 ;


Quote:
[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 ?
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Old 03-05-2015, 01:38 PM   #218
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Quote:
Originally Posted by ClemBuntu View Post
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.
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Old 03-06-2015, 06:37 AM   #219
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Quote:
Originally Posted by fahmida View Post
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
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Old 03-06-2015, 06:43 PM   #220
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Quote:
Originally Posted by ClemBuntu View Post
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.
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