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  • chip-seq read extension (36bp->200bp)

    Hola!

    I was wandering if there's any command line tools (bedtools, samtools) other than R packages, to extend the reads from the base length b/w 36-50 bp to 200bp strand specific. I dont want to use R, as I have to load the file in the environment which is time consuming, I can make awk script easily, but there might be something present already. I want to do it because if I generate a coverage(bedGraph file) directly collapsing the bam file, I get small continuous mountains, which are more or less the referring to single gene target. (Look at the attached picture)

    Also, another question is, does the chip-seq data should be normalized (same number of reads in control and sample) before calling peaks in Macs. For me, the mockIP control always less than 2/3rd the number of reads of control. I did a test, the number of positive peaks went down by 30-40% and -ve peaks came up by 30% after using Macs on normalized data.

    Thanks
    Sukhi
    Attached Files

  • #2
    Most genome browsers don't like it when your reads extend beyond the end of chromosomes which will get a lot more of when you extend your reads. See:
    I’ve been spending my down time this last week looking into ways to streamline my data processing. Basically that has meant learning how to write scripts. This is a little script I just wrote that …

    The second script does exactly what you are asking for.
    --------------
    Ethan

    Comment


    • #3
      Thanks for the reply.
      There is ucsc tool called bedclip which sorts the read off-end of chromosome problem.

      I was tried using your script but I am getting "Unrecognized character \xE2" problems while executing it, I checked it and I think its because of utf encoding problems. Would providing a download link for the original source file, solve the problem!!.

      Also, in this script do reconstruct the bed file after extension because the some reads in coverage will overlap. I am working with bedGraphs not bed. What do you think.

      Eg:
      Chr1 40 50 4
      Chr2 110 150 3


      After adding 150 to both co-ordinates, the reads will overlap and should be summed up (4th column-density), if I am not wrong.

      Thanks

      Comment


      • #4
        I was looking all over for a script that did what bedclip did. Thanks for the heads up. Anyway, it was a good motivation to learn some Perl.

        It seems hosting code on wordpress.com is not trivial. I uploaded the file here:
        --------------
        Ethan

        Comment


        • #5
          It appears there is a UCSC utility that does what you are looking for as well called bedExtendRanges.
          --------------
          Ethan

          Comment


          • #6
            Sorted

            Hey,

            Your perl script works fine for me , but the chromosome off-end clipping seems not to be the perfect one, but bedclip works fine as well.
            I tried using bedExtendRanges but needs some database configuration, so dropped it.
            So, I am currently using your script which extends the reads directionally, then bedClip to clip the off-end chromosome co-ordinates and mergeBed from bedTools to merge the overlapping reads.

            Works good.

            Cheers
            Sukhdeep Singh

            Comment

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