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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