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  • krish91
    Junior Member
    • Apr 2014
    • 3

    #1

    checking the differential expression from .bed files but using cufflinks/cuffdiff, ??

    Hi,
    I have been trying to see the differential expression of mrna seq by using there .bed files from epigenome data.

    I have converted .bed files to .bam using bedtools and i am using cufflinks for obtaining FPKM values and using cuffdiff for checking the differential expression, but both are resulting 0 FPKM values.
    command used:

    cuffdiff -o Homo_sapiens.GRCh37.75.gtf ips_cell_line/gsm706050_s_sorted.bam UW.Fetal_Spinal_Cord/gsm1101679_sorted.bam

    cufflinks -G Homo_sapiens.GRCh37.75.gtf UW.Fetal_Spinal_Cord/gsm1101679_sorted.bam

    version: cufflinks 1.3.0-2ubuntu1
    Last edited by krish91; 04-10-2014, 10:53 PM.
  • krish91
    Junior Member
    • Apr 2014
    • 3

    #2
    I have actually sorted my .sam file obtained from .bam by

    sort -k 3,3 -k 4,4n gsm1101679.sam > gsm1101679.sam.sorted
    even after sorting it i am getting the same error.
    ERROR:
    Error: this SAM file doesn't appear to be correctly sorted!
    current hit is at chr10:60106, last one was at chr1:249235968
    Cufflinks requires that if your file has SQ records in
    the SAM header that they appear in the same order as the chromosomes names
    in the alignments.
    If there are no SQ records in the header, or if the header is missing,
    the alignments must be sorted lexicographically by chromsome
    name and by position.

    can someone give me a clue how to proceed further..

    Comment

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