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Re: [galaxy-user] [galaxy-dev] Tophat output
by Jennifer Jackson 18 Apr '12

18 Apr '12

Hello Jianpeng, The numbers 87 and 93 are the actual maximum lengths of the aligned regions on either side of the junction. If you want to examine your pair-end data statistically, the "NGS: Picard (beta)" tool group has several tool options. However, examining the track at the gene/transcript level for a few well characterized gene bounds is really the best way to understand how the file describes the data. A browser with your tracks loaded (Trackster or UCSC), the text data files, and the Cufflinks manual/FAQ will likely address most of your questions or at least will be a good orientation. The visual portion of this helps a great deal. http://cufflinks.cbcb.umd.edu/faq.html To address the visualization at UCSC, I can point you to their User Guide: http://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html and contact mailing list: http://genome.ucsc.edu/contacts.html Good luck with your project. Please remember to keep questions to the Galaxy team on our mailing lists so that our entire team and community can contribute/benefit, Thanks! Jen Galaxy team -------- Original Message -------- Subject: RE: [galaxy-dev] Tophat output Date: Wed, 18 Apr 2012 15:23:43 +0000 From: Xu, Jianpeng <jianpeng.xu(a)emory.edu> To: Jennifer Jackson <jen(a)bx.psu.edu> Hi Jennifer, In the history, I have the splice junction file, and click it to show the display at UCSC main. Then I click display at UCSC main. It will open the USCS Genome Browser. Since this is the first time for me to visualize the splice junction, can you give me more instructions on how to visualize it with UCSC genome browser ? Thanks, Jianpeng On 4/18/12 7:58 AM, Xu, Jianpeng wrote: > Thanks a lot, Jennifer. It is very useful and helpful. I got the result using Paired-end reads. The read length for both ends is 100 bp. > > chr20 199821 204701 JUNC00000001 17 - 199821 204701 255,0,0 2 87,93 0,4787 > > Since the read length is 100 bp, why the 87, 93 are less than 100 ? > > Below is a sing end read result: > > chr11 60277777 60278396 JUNC00000001 1 + 60277777 60278396 255,0,0 2 22,28 0,591 > > Can you explain a little bit more ? > > Thanks, > > Jianpeng > ________________________________________ > From: Jennifer Jackson [jen(a)bx.psu.edu] > Sent: Wednesday, April 18, 2012 2:56 AM > To: Xu, Jianpeng > Cc: galaxy-dev(a)lists.bx.psu.edu > Subject: Re: [galaxy-dev] Tophat output > > Hello Jianpeng, > > The output files from TopHat are described on the TopHat tool form: > > --- quote --- > Outputs > > Tophat produces two output files: > > junctions -- A UCSC BED track of junctions reported by TopHat. Each > junction consists of two connected BED blocks, where each block is as > long as the maximal overhang of any read spanning the junction. The > score is the number of alignments spanning the junction. > accepted_hits -- A list of read alignments in BAM format. > > Two other possible outputs, depending on the options you choose, are > insertions and deletions, both of which are in BED format. > ------------- > > BED format is described in the Galaxy wiki, which includes links to the > UCSC BED format description (they authored the format). > http://wiki.g2.bx.psu.edu/Learn/Datatypes#Bed > > Two important rules to remember about BED format: > > rule #1: coordinate data is already reported with respect to the (+) strand > > rule #2: "start" is defined as the smallest coordinate, "end" is defined > as the largest coordinate, due to the rule #1. > > BED files have a 0-based, fully-closed, "start" position in data files, > but in browsers the data will display as 1-based. This means you'll need > to add "1" to any "start" coordinate in a .bed file to locate it in a > display application. The two will not and should not match. The "end" > coordinate is also 0-based, but half-open. This will make it appear to > be 1-based for casual users, so it will match between data files and > display applications. > > ------------- > Using the first data row as an example and this information, we can tell > that: > > chr20 199821 204701 JUNC00000001 17 - 199821 204701 255,0,0 2 > 87,93 0,4787 > > * column 5 is 'score', or 'number of alignments spanning the > junction'. In this case, "17" alignments. > > * column 11 is the blockSizes, or 'read maximal overhang' of the > junctions (max alignment length). The first is 87 bases, the second 93 > bases. > > * column 12 is the blockStarts, or 'overhang start' of the junctions > (alignment start). The first is 0, the second 4787 bases. I am fairly > certain that the first is always 0 and the second could be interpreted > as the 'intron' length, but someone please correct me if this is wrong! > > Some calculations can be done with these numbers with respect to the > overall position of the junction already defined in columns 1,2,3 > (chrom, start, end): chr20:199821-204701 (-) that define the location of > the predicted splices, the flanking aligned regions, and the (presumed) > 'intron'. This example is a bit tricky because the alignment is on the > (-) strand, but for most uses it is enough to simply calculate backwards > from the end coordinate to the start. (Consider the end the start, and > the start the end). If this sounds confusing, that's because it is! When > you visualize the data the concept will make more sense and it is > definitely worth learning about. > > Brief explanation: The first start is 0, which literally means that it > starts at the very beginning of the alignment (0-based), which would be > at position chr20, base 204,701, on the (-) strand. This alignment would > continue for 87 bases, then stop. Then the splice would be present. The > second start is at position (204701 - 4787) = 199914 = chr20, base > 199,914, on the (-) strand. This is where the second splice would be > present. This alignment would continue for 93 bases. The places the end > at (199914 - 93) = 199821 = chr20, base 199,821, on the (-) strand. > Which is the same as the reported global junction start position, which > we are considering our "end", because this is a (-) stranded alignment. > And, it all adds up. > > Trackster would be a good place to start for "Visualization (use the top > menu bar link). The dataset can also be saved as a regular .bed file and > loaded as a custom track into the UCSC Genome Browser (If the direct > link is not fully configured yet). > > Hopefully this helps, > > Jen > Galaxy team > > On 4/17/12 7:20 AM, Xu, Jianpeng wrote: >> Hi, >> >> I have installed local galaxy. I used the Tophat to do the RNA-seq >> alignment and got a output file: splice junction in bed format. >> >> I can not understand it clearly. What does the number 17, 14 ... in the >> column 5 mean ? What does the 87,93 mean ? What does the 0, 4787 mean ? >> Can you explain a little bit to me ? Which tool can be used to view this >> file ? >> >> Thanks, >> >> track name=junctions description="TopHat junctions" >> chr20 199821 204701 JUNC00000001 17 - 199821 204701 255,0,0 2 87,93 0,4787 >> chr20 204631 205520 JUNC00000002 14 - 204631 205520 255,0,0 2 96,87 0,802 >> chr20 205428 205775 JUNC00000003 9 - 205428 205775 255,0,0 2 92,91 0,256 >> chr20 205699 205958 JUNC00000004 15 - 205699 205958 255,0,0 2 87,92 0,167 >> chr20 205929 207067 JUNC00000005 31 - 205929 207067 255,0,0 2 95,97 0,1041 >> chr20 206977 207909 JUNC00000006 19 - 206977 207909 255,0,0 2 93,97 0,835 >> chr20 207884 212679 JUNC00000007 15 - 207884 212679 255,0,0 2 87,76 0,4719 >> chr20 207910 218238 JUNC00000008 1 - 207910 218238 255,0,0 2 61,39 0,10289 >> chr20 212628 218293 JUNC00000009 28 - 212628 218293 255,0,0 2 94,94 0,5571 >> >> >> ------------------------------------------------------------------------ >> >> This e-mail message (including any attachments) is for the sole use of >> the intended recipient(s) and may contain confidential and privileged >> information. If the reader of this message is not the intended >> recipient, you are hereby notified that any dissemination, distribution >> or copying of this message (including any attachments) is strictly >> prohibited. >> >> If you have received this message in error, please contact >> the sender by reply e-mail message and destroy all copies of the >> original message (including attachments). >> >> >> ___________________________________________________________ >> Please keep all replies on the list by using "reply all" >> in your mail client. 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 > -- Jennifer Jackson http://galaxyproject.org

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Obtain 3' UTR
by Luciano Cosme 18 Apr '12

18 Apr '12

Howdy, I am working with a genome that is not well annotated and when I tried to obtain the 3'UTR of around 500 genes from Biomart, I only obtain a few (around 20). What would be the easiest way to obtain the 3'UTR using my RNA-seq data (I have 15 samples sequenced)?. I can see the reads on the genomic browser, but I need an automated process. I tried to get the untranscribed gene from Biomart, then the CDS and cDNA and align them, to see the 3'UTR, but still not able get anything after the stop code. Does anyone have experience with it? I also saw that there a lot of genes with "wrong" annotation within the coding region. Thank you. -- *Luciano Cosme* --------------------------------------------- PhD Candidate Texas A&M Entomology Vector Biology Research Group www.lcosme.com 979 845 1885 cosme(a)tamu.edu ---------------------------------------------

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Tophat mapping
by 杨继文 18 Apr '12

18 Apr '12

Hi, After mapping RNA-Seq paired end reads with Tophat, I can see that most of reads fall into the right regions. However, I still can see lots of reads mapped to non-coding region (the locations where the reads are mapped to don't contain exons). I am wondering if these "non-coding reads" will be included when cufflinks calculates transcript/gene expression. Dying to know your opinion. And another question is: how to know the number of reads mapped to a certain exon? Thanks

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Screencasts on screencast.g2.bx.psu.edu ?
by Peter van Heusden 18 Apr '12

18 Apr '12

Hi there I'm trying to download the screencasts referenced in the Galaxy ENCODE paper (Blankenberg et al 2007). They appear to be on screencast.g2.bx.psu.edu, but while that domain resolves, it doesn't respond to HTTP requests. Does anyone know where they are currently available? Thanks, Peter

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Fwd: Create and Transfer Galaxy Page
by Todd Oakley 17 Apr '12

17 Apr '12

Hi all, [Not sure if this is better suited to galaxy-dev or -user, so I'm sending to both]. I want to document some Galaxy analyses and results for a paper I'm submitting - something like the 'Windshield Splatter' supplement: https://main.g2.bx.psu.edu/u/aun1/p/windshield-splatter. My question is - can I create a Galaxy 'Published Page' from my local Galaxy instance/histories, and then transfer that page to the main Galaxy instance? The reason is that I cannot make my local Galaxy instance public, as I am using a campus resource to host our galaxy. If this is possible, how can I do that? If not, any other ideas? Also, are there any tutorials/pages on how to create Published Pages in Galaxy in the first place? Thanks for reading and best wishes, Todd -- *************************************** Todd Oakley, Professor Ecology Evolution and Marine Biology University of California, Santa Barbara Santa Barbara, CA 93106 USA *************************************** Lab Website <http://labs.eemb.ucsb.edu/oakley/todd/> Twitter: @UCSB_OakleyLab *Recent Papers: * * Cnidocyte discharge regulated by opsin and light BMC Biology Paper <http://tinyurl.com/7dajl2q> | Scientific American Write-up <http://blogs.scientificamerican.com/science-sushi/2012/03/05/hydra-watch-wh…> * Sponge Larvae Could be Guided by Cryptochrome J Exp Biol. Paper <http://jeb.biologists.org/content/215/8/ii> | Nature News <http://www.nature.com/nature/journal/v484/n7393/full/484145d.html>

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Re: [galaxy-user] Adapter Clipping tool
by Xianrong Wong 17 Apr '12

17 Apr '12

Dear all, I'm trying to clip adapter sequences off the ends of my sequencing reads. I tested the tool by purposefully adding parts of the adapter sequence or the full adapter sequence to the 3' end. The tools is amazing in detecting these sequences. However, I encountered some problems. On top of detecting my adapter sequence I found that unrelated sequences are sometimes trimmed off at the 3' end. For example the sequence "TATCCACGTGCTC" was trimmed off even though it does not match the adapter sequence GATCCTCGGCCGCGACC at all. Is there a way to increase the fidelity of the tool? Any comments will be appreciated! Thank you! Best Regards, Jose

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ValueError on startup: workaround
by John Norris [jnn] 17 Apr '12

17 Apr '12

Hi, Just installed Galaxy locally and ran "sh run.sh" for the first time. The web page on 127.0.0.1:8080 gave this error: ValueError: expected only letters, got ' en' This was from line 762 in core.py (in /opt/galaxy-dist/eggs/Babel-0.9.4-py2.6.egg/babel) if not lang.isalpha() raise ValueError('expected only letters, got %r' % lang) So, the workaround is to insert a line before: lang = lang.strip() Then it worked :) Cheers, John John Norris Biorenewables Group (IBERS) Aberystwyth University 01970 823 069

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Help!! Tophat paired end reads
by 杨继文 17 Apr '12

17 Apr '12

Hi, I am very confused by my mapping. Please help me figure out what's wrong with my operation. I got Illumina Hiseq 2000 paired end reads (mouse), and I used Tophat to map these reads. After mapping, I used IGV to have a look at the mapping. I can see that some of the reads fall into exons or span exons (splice junction). These reads seem to fit very well. However, I can also see a lot reads mapped to non-coding region. Are these reads from pre-mRNA? or my mapping was wrong? Did anybody have similar experience?? Furthermore, I can see huge enrichment of reads in 3' UTR (much much more than the coding region). Is this normal? Is this caused by the rRNA depletion method ? Looking forward to your reply Jiwen

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Can we change - bwasw command in Galaxy
by shamsher jagat 16 Apr '12

16 Apr '12

I am doing alignments of 454 reads in bwa and was wondering if there is an option to change it to bwasw and use Galaxy web version? Thanks

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How to use collapsed sequence files in mapping and displaying
by Jun Lu 16 Apr '12

16 Apr '12

I found that there is a "collapse" tool under FASTA manipulation, which will significantly shorten mapping time with bowtie with small RNA reads that tend to have many reads of exact length and sequence after clipping adaptors. The tool generates new names for each unique sequence read with a number indicating the number of times (or occurrences) the unique sequence has appeared in the data. The question is, after mapping with Bowtie, how can I regain this "occurrence" information when displaying in Genome Browser? The current setting will only show one mapped read for each unique sequence, no matter how many times this unique sequence has occurred. Should I write a custom code to expand the resulting sam file based on the occurrences? All runs were executed on the galaxy main server. Any suggestion is appreciated. Jun

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