Re: [galaxy-user] Please help me check the quality of the Tophat mapping to reference genome
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 > 50 > 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 > 50 > 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 > 2 > Use Microexon Search > No > > Please help me find out what is wrong with the Tophat. > > Thanks, > > Jianguang > > > > ___ > The Galaxy User list should be used for the discussion of > Galaxy analysis and other features on the public server > at usegalaxy.org. Please keep all replies on the list by > using "reply all" in your mail client. For discussion of > local Galaxy instances and the Galaxy source code, please > use the Galaxy Development list: > >http://lists.bx.psu.edu/listinfo/galaxy-dev > > To manage your subscriptions to this and other Galaxy lists, > please use the interface at: > >http://lists.bx.psu.edu/ > -- Jennifer Jackson http://galaxyproject.org ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
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 50 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 50 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 2 Use Microexon Search No Please help me find out what is wrong with the Tophat. Thanks, Jianguang ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/ -- Jennifer Jackson http://galaxyproject.org ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
[galaxy-user] Please help me check the quality of the Tophat mapping to reference genome
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 50 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 50 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 2 Use Microexon Search No Please help me find out what is wrong with the Tophat. Thanks, Jianguang ___ The Galaxy User list should be used for the discussion of Galaxy analysis and other features on the public server at usegalaxy.org. Please keep all replies on the list by using "reply all" in your mail client. For discussion of local Galaxy instances and the Galaxy source code, please use the Galaxy Development list: http://lists.bx.psu.edu/listinfo/galaxy-dev To manage your subscriptions to this and other Galaxy lists, please use the interface at: http://lists.bx.psu.edu/
