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  • how to get uniquely aligned reads from bowtie

    hi
    i have rum bowtie 2 using —very-sensitive option and got result like this

    Code:
    14559177 reads; of these:
      14559177 (100.00%) were unpaired; of these:
        4317487 (29.65%) aligned 0 times
        7816644 (53.69%) aligned exactly 1 time
        2425046 (16.66%) aligned >1 times
    70.35% overall alignment rate
    i just want to get the uniquely aligned reads alone i,e., 53.69% of reads (78126644). but my output file (sam format) seems to have the those which are aligned>1 times how to get rid of them?

    in previous versions of bowtie i use to use -m option for that

  • #2
    Filter based on having the same read name (1st column) at multiple rows, or based on the mapping quality (5th column).

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    • #3
      filter on the 5th column, only retain reads with a q score of 255. This is the score given to all reads that are uniquely mappable. If you have bam files you can use

      samtools view -q 255 your.bam > your_filtered.sam

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      • #4
        Originally posted by anurupa View Post
        hi
        i have rum bowtie 2 using —very-sensitive option

        i just want to get the uniquely aligned reads alone i,e., 53.69% of reads (78126644). but my output file (sam format) seems to have the those which are aligned>1 times how to get rid of them?

        in previous versions of bowtie i use to use -m option for that
        Bonjour anurupa,

        I'm encountering the same trouble following switching from bowtie1 to bowtie2. Did you find out a satisfying solution ?

        I'm not sure that a filtering based on MAPQ using [samtools view -q] gives the desired result, as a condition for "uniqueness" for a given read is that its MAPQ is high on the best mapped position AND "much" higher than in any other mapped position.

        Mathieu

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        • #5
          Bowtie2 must somehow define what is a unique alignment (may it be related to the MAPQ or not). This definition remains unclear to me.

          Mathieu

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          • #6
            SAM files created by bowtie contains a 'XS:' tag for reporting secondary alignments for a given read.

            You can check that the number of lines containing the 'XS:' tag corresponds to the number of reads showing >1 alignment(s) :
            Code:
            grep 'XS:' your_alignment_file.sam | wc -l
            A working solution consists in removing the lines containing the ':XS' tag using sed :
            Code:
            sed '/XS:/d' your_alignment_file.sam > your_alignment_file_1alignmentonly.sam
            (Thanks go to Christelle, our group's everyday-very-valuable bioinformatician...).

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            • #7
              you are correct MGineste. I have filtered my reads by
              Code:
               grep -v XS:i: my file>output

              Comment


              • #8
                Removing the lines with "XS:" will leave the corresponding headers behind, which could create a problem to SAM/BAM viewers. That happened in my case.

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