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)

Maximum number of mismatches that can appear in the anchor region of spliced alignment

The minimum intron length

The maximum intron length

500000

Allow indel search

Yes

Max insertion length.

Max deletion length.

Maximum number of alignments to be allowed

Minimum intron length that may be found during split-segment (default) search

Maximum intron length that may be found during split-segment (default) search

500000

Number of mismatches allowed in the initial read mapping

Number of mismatches allowed in each segment alignment for reads mapped independently

Minimum length of read segments

Use Own Junctions

Yes

Use Gene Annotation Model

Yes

Gene Model Annotations

iGenome version of mm9 genes. GTF

Use Raw Junctions

Only look for supplied junctions

Use Closure Search

Use Coverage Search

Yes

Minimum intron length that may be found during coverage search

Maximum intron length that may be found during coverage search

20000

Use Microexon Search

Please help me find out what is wrong with the Tophat.

Thanks,

Jianguang