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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