Hi,

The contig is co-linear with itself !

I think that computing the same with v2.0.0-rc7 and then checking the coverage readouts
in RayOutput/BiologicalAbundances/_DeNovoAssembly will shed light on this.

Also, I saw that you used k-mer length of 33. So you must have compiled with
MAXKMERLENGTH=64 or something like that. There was some users that had some
consistency problems when changing MAXKMERLENGTH from the default which is 32.

Maybe this is related. MAXKMERLENGTH=64 does not crash, but there is maybe still
some bugs. And I think I fixed a few of these bugs since v1.7.

v1.7 was post-assemblathon. v2.0.0 has many changes in it, aside from the new RayPlatform.


          Sébastien

Le 2012-05-18 03:46, Mitchell Stanton-Cook a écrit :
Hi Seb,

Sorry about the bombardment.

I thought you would like this. It's a self-self dot plot of the "pseudomolecule" of the concatenated contigs.

Regards

Mitch


On Fri, May 18, 2012 at 3:46 PM, Mitchell Stanton-Cook <m.stantonc...@gmail.com <mailto:m.stantonc...@gmail.com>> wrote:

    Hi Seb,

    We have looked at a few more things while you have been sleeping ;-)

    We re-ran the assemblies to get the .afg files and loaded them
    into hawkeye.

    It appears that somehow Ray duplicates reads. That is, there are
    more reads in the contigs then actually went into the assembly.

    (TotalReads < ReadsInContigs)

    The difference is equal to the SingletonReads value (which is
    negative!)

    Somewhere Ray is artificially duplicating (certain?) reads.

    I did a quick idiot check at my end (although I don't think it
    matters)  to make sure we did not send duplicate reads into Ray
    (i.e. identical4 line fastq block) and that is all OK.

    We have also noticed, that in some contigs there appears to be
    stretches of 0 coverage... We're not to sure of what's going on here.

    Hope this is of use.


    Cheers

    Mitch





    On Fri, May 18, 2012 at 12:32 PM, Mitchell Stanton-Cook
    <m.stantonc...@gmail.com <mailto:m.stantonc...@gmail.com>> wrote:

        Hi Seb,

        Thanks for the reply.


        A bit more background:
        -------------------------------
        This is 100 bp Illumina PE data (insert of ~300 bp s.d of
        about ~10%).

        ~ 1000X coverage. Ray (1.7) did not like this high coverage
        (you have previously commented on the user-list about this).
        We sampled this down to ~100X coverage.

        We also cleaned the reads (you have pointed this is not
        necessary, but having a consistent cleaned set makes
        downstream analysis i.e. snp calling much easier). All input
        bases have a Q score >= 30. Reads > 70bp after trimming were
        filtered. After this the mean reads size is ~97 bp. We only
        consider read pairs (if 1 one of the sequences in the pairs
        fails a cleaning criterion, both do) and hence no single end
        reads go into Ray.

        Ray was executed like this (we used Ray's internal
        estimations/calculations to determine the best parameters):

        mpiexec -n $PROC Ray -i XXXX.fastq -k $K -o $OUT

        We looked at kmers from 15-35 in increments of 2.

        The genome is ~1.8 Mb. From the assemblies it appears there
        are not a lot repetitive elements in the genome. I have
        attached a csv of the results (XXXX_ALL.csv) ( abbreviations:
        c= contigs, s= scaffold, N = scaffolding character).

        Results for kmer 21 and kmer 23 are interesting.

        kmer 21) As previously mentioned we have a 63 Kb duplication.
        For 63 Kb the start of the two contigs are almost identical (3
        mismatches and 1 gap):

          Score = 1.165e+05 bits (63086),  Expect = 0.0

          Identities =63093/63096  <tel:63093%2F63096>  (99%), Gaps = 1/63096 
(0%)
          Strand=Plus/Plus

        I have attached the alignment (XXXX_b2s.aln)

        There is also a smaller 5 Kb duplication detected:

        Score = 9583 bits (5189),  Expect = 0.0

          Identities = 5192/5193 (99%), Gaps = 1/5193 (0%)
          Strand=Plus/Minus


        kmer 23) (Notice there is about 63 Kb difference between the
        "c 100 bp total len" in the .csv file of kmer 21 and kmer 23).
        From the blast2seq there is no such 63 Kb duplication found.

        Now, once again focusing on the "c 100 bp total len"  in the
        .csv file. I propose kmer 21 duplicate, kmer 23 no-duplicate,
        kmer 25 non-duplicate, kmer 27 duplicate (I verified this
        true), kmer 29 non-duplicate. Strangely kmer 31 is missing
        about 200 Kb in comparison to the other assemblies.

        What we are wondering is:

        1) Why is there large, almost identical duplicates present in
        the assemblies?
        2) Why do we see it in some kmers and not others? (I could
        understand if lower kmer = duplicates, higher kmers =
        non-duplicates if these duplicates are propagated by a
        sequencing error)
        3) Is this a bug or an inherit issue with graph based assembly?
        4) If possible, how can we fix this?
        5) How can I help you with this?


        I hope I have provided you with enough information. If not
        please let me know.

        p.s. I have know problem with this going to the list as long
        as the attachments are not included.



        Regards

        Mitch



        On Fri, May 18, 2012 at 12:25 AM, Sébastien Boisvert
        <sebastien.boisver...@ulaval.ca
        <mailto:sebastien.boisver...@ulaval.ca>> wrote:

            Hi,

            Is the 63 kB perfectly duplicated in your assembly ?


            Le 2012-05-17 01:26, Mitchell Stanton-Cook a écrit :

                Hi Seb,

                Hope all is well.

                I was wondering if you have ever seen duplicate
                sequence in contigs.

                We have ~63 kB duplicate at the start of two different
                contigs. Beyond this it's unique.

                This is Ray 1.7.

                I'm re-running with Ray2.0rc5.

                I came across these posts (ABySS specific):

                
http://groups.google.com/group/abyss-users/browse_thread/thread/264e894b4ec0c96d/30dab75afa686878
                
http://groups.google.com/group/abyss-users/browse_thread/thread/7a03ee033b11afc4
                
http://groups.google.com/group/abyss-users/browse_thread/thread/f0f3a650bd12cf1e


                Any ideas?

                Regards

                Mitch






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