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  • #16
    Hi kenietz
    I have read you post and like to ask you about tophat2 commands. Actually, my problem is that I am using mouse RNAseq and run the tophat2 and cufflinks but when I analysed the data in cummeRbund it gives me :
    CuffSet instance with:
    4 samples
    39098 genes
    0 isoforms
    0 TSS
    0 CDS
    0 promoters
    0 splicing
    0 relCDS

    I think my problem is not considering the read length (101 x 2) and insert size 80-380(mean 150).
    How can I solve this problem ? I read your post and saw that you have solved it. May you please let me the solution.
    Thank you very much in advance

    Originally posted by kenietz View Post
    i have a question on that topic.
    I am trying to map some 76x2 Illumina data with tophat-2.0.4. My reads are 76bp and the fragment size is around 280bp. So i choose '-r 128 --mate-std-dev 40' as i have a pdf with graph for the fragment size. I took only 1000 reads and mapped them with tophat. From samtools flagstat i get:

    1622 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    1622 + 0 mapped (100.00%:-nan%)
    1622 + 0 paired in sequencing
    763 + 0 read1
    859 + 0 read2
    656 + 0 properly paired (40.44%:-nan%)
    676 + 0 with itself and mate mapped
    946 + 0 singletons (58.32%:-nan%)
    16 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)

    properly paired only 40%. i ran cufflinks on the accepted.bam and see this:

    Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (
    [15:16:58] Inspecting reads and determining fragment length distribution.
    > Processed 86 loci. [*************************] 100%
    Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges. It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.
    > Map Properties:
    > Normalized Map Mass: 225.85
    > Raw Map Mass: 225.85
    > Fragment Length Distribution: Truncated Gaussian (default)
    > Default Mean: 200
    > Default Std Dev: 80
    [15:16:58] Assembling transcripts and estimating abundances.
    > Processed 86 loci. [*************************] 100%

    Fragment length mean 200 and Std.Dev 80.

    Well i dont get it. Does that mean that my fragment length is actually around 200 and not around 280 as i am told by the graph? And should i use that value for mapping with tophat?

    Because of that result i tried mapping with tophat with -r 48 (200 - 2*76) but that yeilded 31.7% properly paired reads.

    Im a bit confused. Can someone please explain to me what is going on?

    Thank you in advance.


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