Hi Jen,
Thank you very much for your information. I will not worry about the Tophat 
outputs now. For this particular run, I used a single-end dataset. The whole 
experiment contains both paired-end datasets datasets and single-end datasets. 
I ran Tophat with paired-end setting for the paired-end datasets, and 
single-end setting for the single-end datasets. And then ran Cufflink, 
Cuffmerge, and Cuffdiff.
Jianguang

________________________________________
From: Jennifer Jackson [j...@bx.psu.edu]
Sent: Monday, August 27, 2012 12:36 PM
To: Du, Jianguang
Cc: galaxy-user@lists.bx.psu.edu
Subject: Re: [galaxy-user] Please help me check the quality of the Tophat 
mapping to reference genome

Hello Jianguang,

This is the expected output from this particular tool. Your TopHat
output file 'accepted hits' contains only mapped data.

I did notice this option for the TopHat run:
 > Is this library mate-paired?
 > Single-end

Your data was originally paired end - so this is unexpected. But perhaps
you are working with a different dataset(s) now? If you are running with
the original paired dataset, then this is would be an option to correct
- change to mate paired = yes and run TopHat with both the fwd and rev
reads in a single mapping process. (The same method as in the RNA-seq
tutorial).
http://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis-exercise

Best,

Jen
Galaxy team


On 8/27/12 8:15 AM, Du, Jianguang wrote:
> Dear All,
>
> I ran "Flagstat" under "NGS: SAM Tools" to check the quality of the
> Tophat output (the file of accepted hits).  I got the diagnosis results
> as follow:
>
> 9471730 + 0 in total (QC-passed reads + QC-failed reads)
> 0 + 0 duplicates
> 9471730 + 0 mapped (100.00%:-nan%)
> 0 + 0 paired in sequencing
> 0 + 0 read1
> 0 + 0 read2
> 0 + 0 properly paired (-nan%:-nan%)
> 0 + 0 with itself and mate mapped
> 0 + 0 singletons (-nan%:-nan%)
> 0 + 0 with mate mapped to a different chr
> 0 + 0 with mate mapped to a different chr (mapQ>=5)
>
> I ran Tophat with settings as shown below:
>
> Will you select a reference genome from your history or use a built-in
> index?
> Use a built-in index
> Select a reference genome
> /galaxy/data/mm9/bowtie_index/mm9
> Is this library mate-paired?
> Single-end
> TopHat settings to use
> Full parameter list
> Library Type
> FR Unstranded
> Anchor length (at least 3)
> 8
> Maximum number of mismatches that can appear in the anchor region of
> spliced alignment
> 0
> The minimum intron length
> 70
> The maximum intron length
> 500000
> Allow indel search
> Yes
> Max insertion length.
> 3
> Max deletion length.
> 3
> Maximum number of alignments to be allowed
> 20
> Minimum intron length that may be found during split-segment (default)
> search
> 50
> Maximum intron length that may be found during split-segment (default)
> search
> 500000
> Number of mismatches allowed in the initial read mapping
> 1
> Number of mismatches allowed in each segment alignment for reads mapped
> independently
> 1
> Minimum length of read segments
> 25
> Use Own Junctions
> Yes
> Use Gene Annotation Model
> Yes
> Gene Model Annotations
> /iGenome version of mm9 genes. GTF/
> Use Raw Junctions
> No
> Only look for supplied junctions
> No
> Use Closure Search
> No
> Use Coverage Search
> Yes
> Minimum intron length that may be found during coverage search
> 50
> Maximum intron length that may be found during coverage search
> 20000
> Use Microexon Search
> No
>
> Please help me find out what is wrong with the Tophat.
>
> Thanks,
>
> Jianguang
>
>
>
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--
Jennifer Jackson
http://galaxyproject.org
___________________________________________________________
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