Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • NOTEST on Cuffdiff output

    I am new to the Tuxedo Suite and decided to start by going through the workflow in the Nature Protocols publication. All seemed to be going well until I got to the point of differential expression analysis with Cuffdiff. There, the output file gene_exp.diff had NOTEST in the status column for almost all genes. I looked at the Cufflinks manual, which indicated that this output can result from not having enough alignments for testing in any given gene. I then used Samtools to be sure the .bam files that were generated by TopHat had a number of mapped reads that was comparable to the Nature Protocols example. The results, for all accepted_hits.bam files suggested that the number of mapped reads was very similar to what was expected according to the protocol. An example is below:

    $ samtools idxstats C2_R3_thout/accepted_hits.bam

    2L 23011544 4591000 0
    2R 21146708 5029664 0
    3L 24543557 4112826 0
    3R 27905053 5317642 0
    4 1351857 194612 0
    M 19517 0 0
    X 22422827 4157222 0

    I then tried to use the Cuffdiff option -c, to reduce the minimum number of alignments needed at a locus from 10 to 5, but the resulting gene_exp.diff file still had NOTEST in the vast majority of genes.

    In case it helps, the Cuffdiff command that I used was as follows:

    $ cuffdiff -o diff_out -b ./Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa -p 8 -L C1,C2 -u merged_asm/merged.gtf ./C1_R1_thout/accepted_hits.bam,./C1_R2_thout/accepted_hits.bam,./C1_R3_thout/accepted_hits.bam ./C2_R1_thout/accepted_hits.bam,./C2_R2_thout/accepted_hits.bam,./C2_R3_thout/accepted_hits.bam

    If any additional information is necessary for you to help me troubleshoot this issue, please let me know.

  • #2
    I got the similar issue here. Any idea?

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Strategies for Sequencing Challenging Samples
      by seqadmin


      Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
      03-22-2024, 06:39 AM
    • seqadmin
      Techniques and Challenges in Conservation Genomics
      by seqadmin



      The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

      Avian Conservation
      Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
      03-08-2024, 10:41 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, Yesterday, 06:37 PM
    0 responses
    8 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, Yesterday, 06:07 PM
    0 responses
    8 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-22-2024, 10:03 AM
    0 responses
    49 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-21-2024, 07:32 AM
    0 responses
    67 views
    0 likes
    Last Post seqadmin  
    Working...
    X