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  • Lots of false positives with SAMtools SNP discovery

    Hello,

    I simulated a mutated Drosophila Melanogaster genome from the reference one and then I used a sequencer simulator to produce reads on that genome (with potential sequencing errors). I launched BWA to map the reads on the reference and then I used samtools pileup for discovering the SNPs. Since my genome is simulated I can check if the found SNPs are correct.

    The results were quite surprising since samtools found 149,126 SNPs whereas only 19,976 were actually sequenced. Even if samtools found 94.24% of the SNPs, 87.38% of the results were false positives.

    For example on the chromosome 2L, in the 10,000 first bp, I should have 3 SNPs at positions 8405, 9049 and 9290.

    Here is the content of the pileup file produced by samtools for this region:
    Code:
    2L      8117    G       A       33      33      37      2       A$a     ~~
    2L      8118    T       C       30      30      37      1       c$      ~
    2L      8225    C       A       30      30      37      1       ^FA     ~
    2L      8226    C       A       30      30      37      1       A       ~
    2L      8398    A       G       54      54      37      9       GggGGGGgG       ~~~~~~~~~
    2L      8552    A       W       1       1       37      3       T,,     ~~~
    2L      8556    C       S       1       1       37      3       .,g     ~~~
    2L      9049    C       A       66      66      37      13      aaaAAaAAaaAAa   ~~~~~~~~~~~~~
    2L      9051    A       R       8       8       37      13      ,,,G.,..g,..,   ~~~~~~~~~~~~~
    2L      9290    C       A       81      81      37      18      AaaAaAaAAAAaAAAAAa      ~~~~~~~~~~~~~~~~~~
    To produce the pileup file, I launched the following commands:
    Code:
    samtools view -buSt dmel5.fai file.sam | samtools sort - file.sorted
    samtools pileup -vcf dmel5.fa file.sorted.bam > file.pileup
    Am I misinterpreting the pileup file or am I executing samtools in the wrong way?

    I've read that topic. I tried VarScan which doesn't fit my needs. If I understood well, we have to tell it under which criteria a variation has to be considered as a SNP. I also tried mpileup. Surprisingly in the output file I only have indels, it didn't detect any substitution. And once again there are plenty of false positives among these indels.

    If you have any idea of what's wrong, it would be very helpful to tell me

    Thanks in advance!
    Last edited by mikael; 02-15-2011, 08:37 AM.

  • #2
    Two possibilities come to mind:

    - The FASTQ quality letters are always "~", which is a score of 62. Is this supposed to indicate that your sequencer simulator did not simulate any read errors? If it did simulate read errors but indicated perfect base call quality, you can hardly blame samtools for taking the base calls as absolute truth. Note that samtools pileup does take teh quality scores into account, and your putting them to maximum confronts the tool with an unusual situation.

    - Your read aligner may have included low quality or ambiguous mappings without indicating this. Check the alignment quality scores (5th column, I think) in your SAM file. Samtools pileup may expect them to be reliable, but not all aligner set them to proper values.

    Simon

    Comment


    • #3
      Also, you have extremely low coverage at those positions. Like Simon said, if the base quality is high (62) then how can they not be believed give no other evidence (at the low coverage)?

      Comment


      • #4
        Indeed, that's probably coming from the lack of quality information. I'll try to see if the read simulator (Flux Simulator) can produce that.

        Thanks!

        Comment


        • #5
          Hello,

          I am currently using the BAM files (August 2009 release) from the pilot 1 dataset of the 1000 genomes project. I am running the BAM files through samtools and bcftools but I get a high number of SNP calls which includes a high number of false positives. Any idea why this is happening?

          The command line i am using is :

          ./samtools mpileup -C50 -m2 -F0.0001 -Dugf <reference_genome> -r 20:10,000,000-11,000,000 <inds list> > testing.bcf

          ./bcftools/bcftools view -p 0.99 -Gvc testing.bcf > testing.vcf

          Thanks in advance

          Comment

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