- galaxy-user - lists.galaxyproject.org

Online galaxy turning everything red on RNAseq steps
by Elwood Linney 10 Oct '13

10 Oct '13

Hello, I have been waiting a few weeks to process some RNA seq datasets but woke up this morning with lots of steps red. I thought it just might be because of the movement of the system but I processed steps for some histories and everything has turned red. I also noticed that online Galaxy now has RNAseq steps separated into two sections--does this have something to do with the problems? el linney Duke University Medical Center

3 2

Empty bowtie2 output
by Mikel Egaña Aranguren 10 Oct '13

10 Oct '13

Hi; I'm trying to use the bowtie2 wrapper but it generates and empty output with the "no peek" sign. I have sucesfully recreated the analysis with the same datatsets on the shell, so it must be something to do with Galaxy, but I don't know where to look at. Any clues? Thanks I'm using bowtie2-2.1.0 and Galaxy: changeset: 10421:a477486bf18e branch: stable tag: tip user: Nate Coraor <nate(a)bx.psu.edu> date: Thu Sep 26 11:02:58 2013 -0400 summary: Bugfix for tool-to-destination mapping, tool ids are lowercased but the mapping id was not lowercased -- Mikel Egaña Aranguren, Ph.D. http://mikeleganaaranguren.com

2 1

Galaxy available disk space bug
by Itys Comet 08 Oct '13

08 Oct '13

Hi everybody, Despite I purged all data from my Galaxy account and double checked that my account is actually empty by looking at Saved Histories/Advanced Search/status:all, my account is indicating that I am using half of it (as indicated in User/Preferences). Moreover, this is preventing me to actually use more than half of my account showing that it is not a display problem. It is also not a refresh rate problem since this problem started several months ago. Could you please help me with that? All the best, Itys -- Itys COMET, Ph.D. - Helin Group BRIC - University of Copenhagen Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark Phone: +45-353 25602 Fax: +45-353 25669 Email: itys.comet(a)bric.ku.dk Web page: http://www.bric.ku.dk/research/Helin_Group/

2 1

replace function on galaxy
by Xianrong Wong 08 Oct '13

08 Oct '13

Hello, I have been using the compute function in galaxy to replace sequences with delimiters for processing my reads. I realized that the replace "code" in compute no longer works. Is there any other way to replace sequences with delimiters? Jose

2 1

Re: [galaxy-user] Metagenomic filtering
by Scott Tighe 08 Oct '13

08 Oct '13

Jing All good thoughts and if I remember correctly, custom software can indeed to incorparated into Galaxy through use of the "Toolshed" . I'll check into this with Jennifer. Thanks Scott Scott Tighe Senior Core Laboratory Research Staff Advanced Genome Technologies Core University of Vermont Vermont Cancer Center 149 Beaumont ave Health Science Research Facility 303/305 Burlington Vermont 05405 802-656-2557 On 10/6/2013 9:59 AM, Jing Yu wrote: > Dear Scott, > > I think what you propose is doable. > > You may > 1. use a 16s or gyrase DNA sequence as feeds to blast against your > data to get the relative sequences, > 2. and then use the sequences as feeds to blast against your > nucleotide database with appropriate filters. > > There are several ways to make the steps. For example, you may already > have the 16s sequence from assembly against a reference genome. > And for Step 2, if you are not blasting thousands of times a day, and > believe in the recent stability of NCBI, then a simple web_blast code > will do the trick. Otherwise, since the local blast+ toolkit doesn't > provide the equivalent organism filters, you'll have to work a wit bit > on it: > > Make a nucleotide database for Prokaryotes. > Search txid561[ORGN] on http://www.ncbi.nlm.nih.gov/nuccore (this is > for Escherichia as an example), > Send to 'File' -> Format ->GI List > When Blast, use this GI list as the value of this argument: > -negative_gilist > Then parse the Blast result. > > Most of these can be automated with some code, but I don't know how to > incorporate it into Galaxy. > > Regards, > Jing > On 4 Oct 2013, at 23:52, Scott Tighe <scott.tighe(a)uvm.edu > <mailto:scott.tighe@uvm.edu>> wrote: > >> Dear Jing >> >> What you have outlined below is perfect. >> >> I wonder how hard it would be to design a few filters that only look >> a certain genes and or filter model organisms out of the dataset. >> >> For example, say you want only data for 16s or only gyrase, but no >> /E.coli/ and no /Pseudomanas aeroginosa/ >> >> Scott >> Scott Tighe >> Senior Core Laboratory Research Staff >> Advanced Genome Technologies Core >> University of Vermont >> Vermont Cancer Center >> 149 Beaumont ave >> Health Science Research Facility 303/305 >> Burlington Vermont 05405 >> 802-656-2557 >> On 9/25/2013 12:06 AM, Jing Yu wrote: >>> Hi Scott, >>> >>> My first thought is: >>> >>> 1. Remove rDNA sequences (and/or other well known highly-conserved >>> sequences to reduce the workload in step 2). >>> 2. Blast, then remove sequences with > (say 99%) match to > (say 5) >>> genus. (Optional if step 1 is already good enough) >>> >>> >>> For step 1: >>> Build a fasta file of the chosen highly conserved sequences, and >>> use it as a feed to blast against your MiSeq result. >>> Remove positive hits. >>> For step 2: >>> Blast remaining MiSeq sequences against NCBI (or whatever) database. >>> Remove if it hits more than n genus. >>> >>> Jing >>> On 24 Sep 2013, at 22:17, Scott Tighe <scott.tighe(a)uvm.edu >>> <mailto:scott.tighe@uvm.edu>> wrote: >>> >>>> Jing et al >>>> >>>> Thank you for the offer to write some code to help advance the >>>> metagenomics arena. It is certainly needed. >>>> >>>> So the problem is well known with megablast and shotgun >>>> metagenomics and without proper understanding and correct software >>>> will yield very misleading and in many cases incorrect data. For >>>> those of us who wish NOT to move to a protein level of comparison >>>> for specific reasons, we are stuck. >>>> >>>> *The Problem:* >>>> >>>> If I megablast 50 million sequences from a HiSeq run, millions of >>>> rRNA sequences will have a 99% match to all microbes rRNA genbank >>>> deposits. Not surprizing since the rRNA is highly conserved. The >>>> difference between E.coli and Shigella is 1 to 2 bases for the full >>>> 1540 bp 16s. So 16s is not useful for Genus level, and certainly >>>> not Species >>>> >>>> *So what happens:* >>>> >>>> The returned matches will have many hits to whatever model organism >>>> is in Genbank. For example E coli has 13000 entries for rRNA and >>>> Sphearotilus has 3 entries for rRNA. If the blasted sequence >>>> matches both, the results will mislead the investigator to think >>>> they have 13000 hits to E coli, EVEN if the microbe is Sphearotilus. >>>> >>>> *The cure?:* >>>> >>>> If there was a way to filter/ remove all hits ? Let say, for >>>> example, that a result has a first match (say E. coli) at >99% a >>>> second match (say Pseudomanas) at >99% and a third , forth and >>>> fifth match >99 for three other organisms. This sequence _must_ be >>>> discarded because it is a conserve sequence. >>>> >>>> Basically conserved sequence is the enemy and invalidates the >>>> entire result. >>>> * >>>> **Another problem:* >>>> >>>> If you have a reference sample with 19 non-model microbes, and you >>>> run that by HiSeq Shotgun for metagenomics and then megablast, what >>>> do you think you get? If E coli is not in the reference sample, >>>> how many hits do you think you get? Yes, 10,000 of thousands. So >>>> without removing conserved sequences, your data is wrong and you >>>> are much better served by culturing and running a Biolog metabolic >>>> panel and comparing to the sequence result. >>>> >>>> So where do we start? I have some shotgun metagenomics data from >>>> the reference sample which included the 19 microbes. That was data >>>> from a MiSeq. >>>> >>>> Scott >>>> Scott Tighe >>>> Senior Core Laboratory Research Staff >>>> Advanced Genome Technologies Core >>>> University of Vermont >>>> Vermont Cancer Center >>>> 149 Beaumont ave >>>> Health Science Research Facility 303/305 >>>> Burlington Vermont 05405 >>>> 802-656-2557 >>>> On 9/20/2013 9:17 PM, Jing Yu wrote: >>>>> Hi Scott, >>>>> >>>>> I can do some perl programming, such as local/remote blasting. Can >>>>> you specify your problem a little bit clearer, so that maybe I can >>>>> write a program to do just that? >>>>> >>>>> Regards, >>>>> Jing >>>>> >>>>> >>>>> >>>>> >>>>> Gerald >>>>> >>>>> 16s is basically useless for identification to genus. Since I >>>>> started sequencing 16s in 1992, I have come to realize that >>>>> without sequencing the full 1540 bases, it is generally >>>>> misleading, and even than, it is not accurate enough to nail genus >>>>> on more than 1/2 the cases. However, what is your feeling on >>>>> ITS and gyrase, They seem to be far more discriminating but those >>>>> databases have been decommissioned sometime ago. >>>>> >>>>> The desirable thing would be that Galaxy or NCBI add a "filter >>>>> conserved genes" [ ie any hit with a second choice greater than 3% >>>>> distance]. Something such as that. >>>>> >>>>> If you (or others) are aware of such a thing, I'd love the here >>>>> about it. >>>>> >>>>> Sincerely >>>>> Scott >>>> >>> >> >

1 0

How to lauch bulit cluster on Cloudman?
by Yan He 08 Oct '13

08 Oct '13

Dear all, I just established Galaxy Cloudman on Amazon EC2. I created a cluster named "exon_capture" and uploaded a lot of data to it. After some analysis, I terminated the cluster. The second time I wanted to get access to the "exon_capture" cluster, I created a new instance under the "exon_capture". However, under this instance, the "access galaxy" button is in grey and not active. I tried several time, but the button was always in grey. Does anyone know what's wrong? Did I miss something? It may be a very simple question, but it bothers me a whole afternoon. Thanks a lot! Yan

2 1

Adding a tool to Galaxy Main Server
by Hamid Reza Hassanzadeh 08 Oct '13

08 Oct '13

Hi everybody, We are going to add our gene prediction tool to Galaxy Main server, anybody know whom we should contact? Thanks -- Hamid Reza Hassanzadeh, PhD Student & Graduate Research Assistant, Bioinformatics Lab, Center for Bioinformatics and Computational Genomics Joint Georgia Tech and Emory Wallace H Coulter Department of Biomedical Engineering, Department of Computer Science at Georgia Institute of Technology office: KACB 1343

2 1

Fwd: Exome Sequencing Analysis
by Johnathan Cooper-Knock 08 Oct '13

08 Oct '13

Hello, My name is Johnathan Cooper-Knock, I am a clinical fellow based at the University of Sheffield, UK. I am trying to use Galaxy for analysis of DNA sequencing data and I have run into a problem. I am trying to run the SAM/BAM Hybrid Selection Metrics step on Galaxy (part of the Picard tools) but I can't find bait and target bed files that Galaxy will accept. My library capture was performed using the Aglilent All Exon V4 kit and I have uploaded the 'S03723314_Regions.bed' and 'S03723314_Covered.bed' files from ' https://earray.chem.agilent.com/suredesign/search.htm' but Galaxy does not even seem to recognise them as options for entry. Is this a formatting issue? Do you know where I can get ready formatted files? Thanks Johnathan

2 1

galaxy cloud: location of imported and converted fastq's
by p Vedell 07 Oct '13

07 Oct '13

Hi, I had fastq.gz files in an Amazon S3 bucket. I created a Galaxy on the Cloud instance using CloudMan. I used GetData and pasted in the url's of the fastq.gz files into Galaxy on the Cloud. They were successfully imported into the session and converted to fastq's, it seems. However, at the next step (fastq groomer), Galaxy on the Cloud appears to expect that the imported and converted fastq files are in the same S3 bucket where the fastq.gz files were. But, they are not there. I read somewhere in documentation or notes that the data is actually in /mnt/galaxyData folder. However, I am not sure how to point fastq groomer to this place in the web interface or alternatively, I am not sure how to move the fastq's back to the s3 bucket (w/o downloading and re-uploading which would be very time-consuming). Thanks for any help you can provide. PT

2 1

Re: [galaxy-user] Metagenomic filtering
by Scott Tighe 06 Oct '13

06 Oct '13

Dear Jing What you have outlined below is perfect. I wonder how hard it would be to design a few filters that only look a certain genes and or filter model organisms out of the dataset. For example, say you want only data for 16s or only gyrase, but no /E.coli/ and no /Pseudomanas aeroginosa/ Scott Scott Tighe Senior Core Laboratory Research Staff Advanced Genome Technologies Core University of Vermont Vermont Cancer Center 149 Beaumont ave Health Science Research Facility 303/305 Burlington Vermont 05405 802-656-2557 On 9/25/2013 12:06 AM, Jing Yu wrote: > Hi Scott, > > My first thought is: > > 1. Remove rDNA sequences (and/or other well known highly-conserved > sequences to reduce the workload in step 2). > 2. Blast, then remove sequences with > (say 99%) match to > (say 5) > genus. (Optional if step 1 is already good enough) > > > For step 1: > Build a fasta file of the chosen highly conserved sequences, and > use it as a feed to blast against your MiSeq result. > Remove positive hits. > For step 2: > Blast remaining MiSeq sequences against NCBI (or whatever) database. > Remove if it hits more than n genus. > > Jing > On 24 Sep 2013, at 22:17, Scott Tighe <scott.tighe(a)uvm.edu > <mailto:scott.tighe@uvm.edu>> wrote: > >> Jing et al >> >> Thank you for the offer to write some code to help advance the >> metagenomics arena. It is certainly needed. >> >> So the problem is well known with megablast and shotgun metagenomics >> and without proper understanding and correct software will yield very >> misleading and in many cases incorrect data. For those of us who wish >> NOT to move to a protein level of comparison for specific reasons, we >> are stuck. >> >> *The Problem:* >> >> If I megablast 50 million sequences from a HiSeq run, millions of >> rRNA sequences will have a 99% match to all microbes rRNA genbank >> deposits. Not surprizing since the rRNA is highly conserved. The >> difference between E.coli and Shigella is 1 to 2 bases for the full >> 1540 bp 16s. So 16s is not useful for Genus level, and certainly not >> Species >> >> *So what happens:* >> >> The returned matches will have many hits to whatever model organism >> is in Genbank. For example E coli has 13000 entries for rRNA and >> Sphearotilus has 3 entries for rRNA. If the blasted sequence matches >> both, the results will mislead the investigator to think they have >> 13000 hits to E coli, EVEN if the microbe is Sphearotilus. >> >> *The cure?:* >> >> If there was a way to filter/ remove all hits ? Let say, for example, >> that a result has a first match (say E. coli) at >99% a second match >> (say Pseudomanas) at >99% and a third , forth and fifth match >99 for >> three other organisms. This sequence _must_ be discarded because it >> is a conserve sequence. >> >> Basically conserved sequence is the enemy and invalidates the entire >> result. >> * >> **Another problem:* >> >> If you have a reference sample with 19 non-model microbes, and you >> run that by HiSeq Shotgun for metagenomics and then megablast, what >> do you think you get? If E coli is not in the reference sample, how >> many hits do you think you get? Yes, 10,000 of thousands. So without >> removing conserved sequences, your data is wrong and you are much >> better served by culturing and running a Biolog metabolic panel and >> comparing to the sequence result. >> >> So where do we start? I have some shotgun metagenomics data from the >> reference sample which included the 19 microbes. That was data from a >> MiSeq. >> >> Scott >> Scott Tighe >> Senior Core Laboratory Research Staff >> Advanced Genome Technologies Core >> University of Vermont >> Vermont Cancer Center >> 149 Beaumont ave >> Health Science Research Facility 303/305 >> Burlington Vermont 05405 >> 802-656-2557 >> On 9/20/2013 9:17 PM, Jing Yu wrote: >>> Hi Scott, >>> >>> I can do some perl programming, such as local/remote blasting. Can >>> you specify your problem a little bit clearer, so that maybe I can >>> write a program to do just that? >>> >>> Regards, >>> Jing >>> >>> >>> >>> >>> Gerald >>> >>> 16s is basically useless for identification to genus. Since I >>> started sequencing 16s in 1992, I have come to realize that without >>> sequencing the full 1540 bases, it is generally misleading, and >>> even than, it is not accurate enough to nail genus on more than 1/2 >>> the cases. However, what is your feeling on ITS and gyrase, They >>> seem to be far more discriminating but those databases have been >>> decommissioned sometime ago. >>> >>> The desirable thing would be that Galaxy or NCBI add a "filter >>> conserved genes" [ ie any hit with a second choice greater than 3% >>> distance]. Something such as that. >>> >>> If you (or others) are aware of such a thing, I'd love the here >>> about it. >>> >>> Sincerely >>> Scott >> >

2 1