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  • 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.
    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


    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

    Comment


    • 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

      Comment


      • 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?

        Comment


        • 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.

          Comment


          • 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.

            Comment


            • 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

              Comment


              • 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 :
                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 ?

                Comment


                • 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.

                  Comment


                  • 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

                    Comment


                    • 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.

                      Comment


                      • 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?

                        Comment


                        • Originally posted by illinu View Post
                          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

                          Comment


                          • 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

                            Comment


                            • Hello, see if this helps:
                              Code:
                              sudo cpan
                              install Perl4::CoreLibs

                              Comment


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

                                Comment

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