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  • chrom file format problem with bedtools genomecov

    Hi,
    I am new to bioinformatics. I am trying to use the genomecov in the bedtools into bedgraph, which I want to load them onto genome browser. Overal it works perfect with ChIP-seq data, which is non-stranded. However, when I try to do it with HITS-CLIP or RNA-seq, which are strand-specific, it shows the error message
    *****ERROR: Unrecognized parameter: NC10.chrom *****
    I looked into the chrom file, which is like this
    supercont10.1 9798893
    supercont10.2 4478683
    supercont10.3 5274802
    supercont10.4 6000761
    supercont10.5 6436246
    supercont10.6 4218384
    supercont10.7 4255303
    supercont10.8 192308
    supercont10.9 142473
    supercont10.10 125404
    supercont10.11 31696
    supercont10.12 19714
    supercont10.13 13515
    supercont10.14 11565
    supercont10.15 9397
    supercont10.16 8983
    supercont10.17 6701
    supercont10.18 6309
    supercont10.19 4755
    supercont10.20 1646

    I don't know why when I added the option "-strand" then the program refuse to recognize the chorm file, though genomecov does accept it with the sample bam file when "-strand" are deleted.
    Any suggestion are greatly appreciated!!

  • #2
    just add more information for troubleshooting

    I attached all the processes I did from begining for the HITS-CLIP

    tophat -p 8 -G NC10_transcripts.gff3 -o wt_output --library-type=fr-secondstrand <genome_indexes> wt.fq

    next, I sort the bam and calculate the coverage. the command is

    samtool sort accepted_hits.bam sorted.bam
    genomeCoverageBed -ibam <sorted.bam> -strand -bg -g NC10.chrom > output.bg

    then I got the error message

    *****ERROR: Unrecognized parameter: NC10.chrom *****

    Comment


    • #3
      You are use the wrong command line order:
      genomeCoverageBed -ibam -strand -bg -i <sorted.bam> -g NC10.chrom

      Comment


      • #4
        It work now. But patially right. My bam file is acutally strand-specific pair-end sequencing. After calculate the coverage from each strand and merge them, I found that all gene has antisense transcripts, which should be wrong. I guess the genomecov might also count the mate of read from the opposite strand. Do you know how to fix this? Thanks!

        Comment

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