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  • #16
    i also have my problems with oases and it is still kind of a black box because there is no paper about it. If you want less Ns you could probably just reduce the insert length. Of course this is the tricky way and probably not that reasonable. ;-)

    Let me know when you figured out how exactly oases does scaffolding. I tried to get some information out of the code, sad it is not easy to "read" .

    if you have a near relative where the proteome is known you could try out this sort of scaffolding: http://genome.cshlp.org/content/20/10/1432 .

    What you could try for expression level is mapping back the velvet contigs which are used for oases, since these have the coverage in there ID you will also get and idea about the expression levels.

    Comment


    • #17
      ad problem with solid

      [second edit]
      [third edit: seems to work fine. Used BLAT to check the contigs and transcripts - results as expected. However - the difference between asid -merge and the postprocessor script are still unknown.]

      should work:

      sample: arbitrary samplename
      workdir: directory with everything inside

      ### run the preprocessor. In case you want to filter something, do it before this step... (before i pipetted directly into velvet with a filter - reason for the previous mess)

      perl denovo_preprocessor_solid_v2.2.1.pl -run fragment -f3 sample.csfasta -dir workdir/


      ### run velvet. The -amos_file yes option is only required if you use the denovo_postprocessor_solid_v1.6.pl script

      velveth_de workdir 31 -short -fasta workdir/de_fragment_input.de
      velvetg_de workdir -read_trkg yes -amos_file yes


      ### if you want the velvet contigs in nucleotide space: either you use the script and asid_light:

      perl denovo_postprocessor_solid_v1.6.pl --afgfile workdir/velvet_asm.afg --csfasta workdir/cs_fragment_input.csfasta --output workdir/color_reads.ma
      asid_light -convert workdir/color_reads.ma [number of characters per line] > workdir/nt_contigs.fa


      ### or only asid_light:

      asid_light -merge workdir/LastGraph workdir/contigs.fa workdir/cs_fragment_input.csfasta workdir/cs_fragment_input.idx workdir/gap_reads/ workdir/color_reads.ma workdir/asid_scaffolds.de graph2ma [mincontiglength] [library type]
      asid_light -convert workdir/color_reads.ma [number of characters per line] > workdir/nt_contigs.fa

      comparing the output after running velvet only (using the script and asid or only asid) I saw that they are different - reason?
      partly due to min contig length of 80 in asid_light -merge; but there must be another reason...
      already the color_reads.ma files are slightly different - and the contigs seem to be somehow similar - but they are definitively not equal. also runtimes are very different...

      any ideas?

      haven't got the time to look at it yet in detail... update may follow


      [third edit - adding the oases stuff]

      ### run oases (-amos_file yes is required for the script)

      oases_de workdir [whatever options] -amos_file yes


      ### again - to convert the transcripts, use either the script and asid_light:

      perl denovo_postprocessor_solid_v1.6.pl --afgfile workdir/oases_asm.afg --csfasta workdir/cs_fragment_input.csfasta --output workdir/color_reads.ma
      asid_light -convert workdir/color_reads.ma [number of characters per line] > workdir/nt_transcripts.fa

      ### or only asid_light:

      asid_light -merge workdir/LastGraph workdir/transcripts.fa workdir/cs_fragment_input.csfasta workdir/cs_fragment_input.idx workdir/gap_reads/ workdir/color_reads.ma workdir/asid_scaffolds.de graph2ma [mincontiglength] [library type]
      asid_light -convert workdir/color_reads.ma [number of characters per line] > workdir/nt_transcripts.fa

      ## done
      Last edited by schmima; 03-06-2011, 11:52 PM.

      Comment


      • #18
        Oases: segmentation fault

        Hi all,

        I'm trying to use Oases to do a de novo transcriptome assembly of single-end Illumina reads, but am getting the following error message:

        [0.000000] Reading graph file velvet_assembly//Graph2
        [0.001334] Graph has 120201 nodes and 22312981 sequences
        Segmentation fault


        I am using Oases 0.1.18 and Velvet 1.0.18. I assembled the single reads with Velvet 1.0.18 and kept -read_trkg on.

        The size of my Velvet output files are:
        37M contigs.fa
        491M Graph2
        491M LastGraph
        611 Log
        76M PreGraph
        1009M Roadmaps
        3.2G Sequences
        10M stats.txt

        I am trying to do the job on a 128 GB RAM machine.

        Appreciate any suggestions for a solution. Thanks!

        Comment


        • #19
          hm - sounds like an uncatched/unexpected error.

          any errors during the compilation / did you forward all the velvet-settings while compiling oases? eg:

          velvet compilation:
          make 'CATEGORIES=1' 'MAXKMERLENGTH=31'
          oases compilation:
          make 'VELVET_DIR=/blabla/velvet_1.0.18' 'MAXKMERLENGTH=31' 'CATEGORIES=1')

          [I dont think that RAM is a problem... In case the reads are not a total crap it should be no problem - filtered in my case the low quality and low complexity reads. with 20 mio reads (in color space however) I was doing well with my 12 GB desktop computer (k = 31)]
          Last edited by schmima; 03-06-2011, 11:49 PM.

          Comment


          • #20
            if you can reproduce this error, you should tell zerbino about that: http://www.ebi.ac.uk/~zerbino/oases/ join the mailinglist. ;-) zerbino is active there and you get a response quite fast.

            not much you can do when you get "segmentation fault" as an error, what kmer do you use? Probably try again with a higher hash value?

            Comment


            • #21
              Thanks for your suggestions re. Oases: segmentation fault.

              I have recompiled Oases, making sure to give the correct path to Velvet and specifying the same 'CATEGORIES=30' 'MAXKMERLENGTH=99' as for Velvet.

              Unfortunately, I still get the same error. So, this error is indeed very reproducible .

              I'm using a hash value of 57, which gave me the best N50 and largest contig out of a range of 51-99mers - from VelvetOptimiser and subsequent optimisation runs.

              Comment


              • #22
                woah! how many reads you have and how long are they? Never choose a higher hash value then your minimum read length.

                so you also get sementation fault with lower kmers like 43 or higher onces like 76?

                Comment


                • #23
                  I have total of a little over 22 million 99-nucleotide reads. This is from four lanes of Illumina data.

                  I have just assembled one lane of data (5,522,680) and Oases seems to be able to cope with this, at a hash value of 57. So I guess the quantity of input data is a factor for the segmentation fault. I'll raise this with Marcel or Daniel.

                  Thanks all.

                  Comment


                  • #24
                    dear all,
                    I have a problem with OASES. I run this :
                    cd ~/processing
                    velveth 25march/Oidium_fasta_RNAseq/assembly 25 -fasta -shortPaired 25march/Oidium_fasta_RNAseq/Oidium.fa
                    velvetg 25march/Oidium_fasta_RNAseq/assembly -read_trkg yes
                    oases 25march/Oidium_fasta_RNAseq/assembly -alignments yes -unused_reads yes

                    the problem is that wen I add some options to OASES (-alignments yes -unused_reads yes) it does not work at all.

                    Any suggestions?

                    Luigi

                    Comment


                    • #25
                      you need to give the insert length as parameter (-ins_length) as stated in the readme.

                      Which versions of velvet and oases you use?

                      did you compile oases with the same maxkmerlength and categories as used in velvet?

                      Comment


                      • #26
                        try to use the options also during velvetg:

                        velvetg ... -alignments yes -unused_reads yes -read_trkg yes

                        Comment


                        • #27
                          dear Thorondor,
                          In velveth the parameters are CATEGORIES = 2 MAXKMERLENGTH = 31 which are the standard from the Makefile. In Oases I can not trace them back but the Makefile has the some parameters in it. I assume they are the same than.
                          About the answer of Schmima, it is possible to have these files in velvetg but the results can be different between the oases and velvetg.
                          any other suggestions?

                          Luigi

                          Comment


                          • #28
                            yes - the output files normally differ. Just thought that oases may need the velvet output from these files and modifies them (?).

                            btw - what are the errors you get? or in what way is it not working?

                            Comment


                            • #29
                              just call ./oases without parameters and the output will be like:

                              oases - De novo transcriptome assembler for the Velvet package
                              Version 0.1.20

                              Copyright 2009,2010 Daniel Zerbino ([email protected])
                              This is free software; see the source for copying conditions. There is NO
                              warranty; not even for MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.

                              Compilation settings:
                              CATEGORIES = 27
                              MAXKMERLENGTH = 61
                              plus parameters you can set. same works for velveth or velvetg.

                              So you have the newest versions installed of velvet and oases and you have set the insert length only in oases?
                              Don't set any other parameters in velvetg then -read_trkg yes!

                              what files are there in you folder after running velveth and velvetg?

                              Comment


                              • #30
                                Dear Oases users,

                                I'm trying to compile Oases on a Ubuntu 10.10 virtual machine on our server. Directly after installing the OS, i just installed G++ for compiling. Velvet was compiled with no errors, but Oases gives me the following output:
                                Code:
                                [...]
                                gcc -Wall -O3 -D MAXKMERLENGTH=35 -D CATEGORIES=2 -c src/transcript.c -o obj/transcript.o -I/home/jpreuss/Velvet/src
                                src/transcript.c:564: error: static declaration of ‘getTotalCoverage’ follows non-static declaration
                                /home/jpreuss/Velvet/src/graph.h:99: note: previous declaration of ‘getTotalCoverage’ was here
                                make: *** [obj/transcript.o] Error 1
                                Has anyone an idea why this error shows up, or what it means? I got Oases already running on Ubuntu 10.10, perhaps I'm missing another lib?

                                Thanks for any suggestions,
                                Jenzo
                                Last edited by Jenzo; 04-19-2011, 11:07 PM. Reason: Problem solved, see the next posts

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