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  • super0925
    Senior Member
    • Feb 2014
    • 206

    #1

    How to get the expression level of specific gene without annotation in RNA-Seq?

    I have one samples in fastq files from bovine (bovine_sample1.fastq). If I want to see whether a sequence (supposed named "GQ2") is expressed in bovine cells. (I have got the FASTA of this sequence) The sequence is unannotated in the current bovine genome, so might not have been tested in the analyses thus far.
    As we know , in RNA-Seq, the expression level is number of raw reads counts mapped to genome.so my solution is like this:

    My solution (2 steps):
    (1) generate the bowtie2 index of GQ2.

    >GQ2
    ATGGAGCACTTTCCCCGCTGTGTGCACGAGTCCTGGGGTTCCTCAAAGGA
    GCCCCAGAAAACAGAGGTGCTGCAACTCTTGAGCTTAGCGGACCCTGAGG
    .....
    mkdir GQ2Bowtie2Index
    cd GQ2Bowtie2Index
    bowtie2 GQ2.fa GQ2
    ls

    GQ2.1.bt2 GQ2.2.bt2 GQ2.3.bt2 GQ2.4.bt2 GQ2.fa GQ2.rev.1.bt2 GQ2.rev.2.bt2

    (2) map the reads file on to 'GQ2' genome

    bowtie2 --local --very-sensitive-local -p 8 -x ./GQ2Bowtie2Index/GQ2 -U bovine_sample1.fastq

    But when I did it, I got the error.
    After I ran the bowtie2, it goes to the endless loop (obviously it is error). If I terminate the bowtie2, I found the error.
    "bowtie2-align died with signal 2 (INT)"

    Could you please tell me:

    1. Is my solution correct? If it is not, do you have any solution?
    2. If mine is correct, why did I get the error? Do my command lines have the problem? ?
    Last edited by super0925; 07-31-2014, 05:29 AM.
  • Mike2188
    Member
    • Oct 2013
    • 27

    #2
    This may be stupid, even if it is one gene, if you are aligning a transcript sequence, shouldn't you use tophat?

    I couldn't see an error in what you did.

    Comment

    • super0925
      Senior Member
      • Feb 2014
      • 206

      #3
      Originally posted by Mike2188 View Post
      This may be stupid, even if it is one gene, if you are aligning a transcript sequence, shouldn't you use tophat?

      I couldn't see an error in what you did.
      Hi I am sorry I didn't get your meaning clearly.
      Tophat need .gtf annotation files. I don't have gtf file of GQ2 gene.
      I only have FASTA sequence of GQ2.
      Could you give me your suggestion more detail?

      Comment

      • Mike2188
        Member
        • Oct 2013
        • 27

        #4
        Starting files:
        gq2-sequence.fa
        transcripts.fa

        Build Genome Index:
        bowtie2-build gq2-sequence.fa gq2

        Align using tophat and built index:
        tophat2 --b2-very-senstive -p 8 gq2 transcripts.fa
        Last edited by Mike2188; 07-31-2014, 07:36 AM.

        Comment

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