[jp.]

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

Hi,
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 (http://cufflinks.cbcb.umd.edu).
[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.