On Apr 5, 2011, at 3:11 PM, Mike Dufault wrote:
> Hi Anton,
>
> The conditions are give below. Currently, I don't have access to the AWS cloud so I can not share my history at the moment.
>
> Select dataset:
>
>
> which contains:
> Pileup with ten columns (with consensus)
>
> See "Types of pileup datasets" below for examples
> Do not consider read bases with quality lower than:
> 20
>
> No variants with quality below this value will be reported
> Do not report positions with coverage lower than:
> 3
>
> Pileup lines with coverage lower than this value will be skipped
>
> Only report variants?:
> Yes
> See "Examples 1 and 2" below for explanation
>
> Convert coordinates to intervals?:
> Yes
> See "Output format" below for explanation
>
> Print total number of differences?:
> No
> See "Example 3" below for explanation
>
> Print quality and base string?:
> No
> See "Example 4" below for explanation
>
>
> I did save the output from step 15 to my USB stick and I have provided a bit of it below for what it is worth.
>
> chr1 100316588 100316589 A G 255 255 60 141 0 0 137 0 137
> chr1 100575932 100575933 G A 255 255 60 89 89 0 0 0 89
> chr1 100617886 100617887 C T 255 255 60 113 0 0 0 111 111
> chr1 100672059 100672060 T C 255 255 60 225 1 220 0 0 221
> chr1 101203826 101203827 G A 255 255 60 106 105 0 0 0 105
> chr1 103461507 103461508 T A 255 255 60 87 82 0 0 0 82
> chr1 104166495 104166496 T C 255 255 60 168 0 157 0 5 162
> chr1 104256477 104256478 T A 255 255 60 84 82 0 0 0 82
>
> Thanks for your help!
> Mike
>
> --- On Tue, 4/5/11, Anton Nekrutenko <
anton@bx.psu.edu> wrote:
>
> From: Anton Nekrutenko <
anton@bx.psu.edu>
> Subject: Re: [galaxy-user] Analyzing Targeted Resequencing data with Galaxy
> To: "Mike Dufault" <
dufaultm@yahoo.com>
> Cc: "Lali" <
laurafe@gmail.com>, "galaxy-user" <
galaxy-user@lists.bx.psu.edu>
> Date: Tuesday, April 5, 2011, 2:33 PM
>
> Mike:
>
> Which parameters did you use at step 13 (if you used main site to perform these analyses you can share your history with me).
>
> Thanks,
>
> anton
>
>
> On Apr 5, 2011, at 2:22 PM, Mike Dufault wrote:
>
>> Hi all,
>>
>> Like many people on this e-mail chain, I have been looking for advice on how to process Exome data. Below, I have described in detail what I have done with the hope of getting some clarification. Hopefully it will be helpful to many of us!
>>
>> I have SureSelect Exome captured data. The data was delivered to me as two separate files (/1) & (/2). Each file has ~33 million reads; 7.2 GB each. I am looking for SNPs from a family with cancer. Eventually I plan to compare the date from multiple members of the same family to find a related disease SNP.
>>
>> Below is the workflow that I used to process my data. I adapted it from the Screencast titles: "Mapping Illumina Reads: Paired Ends Example." I used all of the same default parameters as in the screencast.
>>
>> At the end of step 13, I had ~4,700,000 SNPs. This seemed like a lot so in step 14, I filtered on column 7 (c7) which I believe is the Quality SNP value. I set the filter as C7>=1 to remove all of the 0 (zero) values for Quality SNP. I figured that if they have a value of zero, they must not be real SNPs. This left me with ~180,000 SNPs.
>>
>> 1: Get Data: Illumina 1.3+ file (/1)
>> 2: Get Data: Illumina 1.3+ file (/2)
>> 3: FASTQ Groomer on data 1
>> 4: FASTQ Groomer on data 2
>> 5: FASTQ Summary Statistics on data 3
>> 6: FASTQ Summary Statistics on data 4
>> 7: Box plot on data 5
>> 8: Box plot on data 6
>> 9: Map with Bowtie for Illumina on data 4 and data 3: mapped reads
>> 10: Filter Sam on data 9
>> 11: SAM-to-BAM on data 10: converted to BAM
>> 12: Generate pileup on data 11: converted pileup
>> 13: Filter pileup on data 12
>> 14: Filter data on 13 (c7>=1)
>> 15: Sort on data 15 (C7; descending order)
>>
>> First, if anyone has ideas on how to improve the workflow, I would be open to suggestions; especially from people experienced with Galaxy.
>>
>> Second, I am concerned that many/most of the SNPs are known. Should I filter my data against the known SNPdb? If so, how can I do this in Galaxy (in Bowtie?)
>>
>> Third, as suggested in the screencast, I did not trim or filter my FASTQ Groomed data because I was interested in SNPs and I could filter on Quality later in the workflow. Would implementing a filtering step on phred quality (~20) at this step save me the step of filtering later on. Currently it takes multiple hours (~16) to process the data from start to finish, would filtering at this step reduce the amount of time that it takes to process my data? Presumably, there would be less data to process. I do this on the AWS Cloud and time is money!
>>
>> Fifth, when using Galaxy on the AWS cloud, does adding additional cores or adding High CPU ( or both) shorten the time to process the data? When I set up extra cores, it appeared that some of them are idle and I don't want to pay for idle cores. If anyone could share information on how best to manage the cloud, it would be appreciated.
>>
>> Finally, what is the difference between “stopping” an instance and “terminating” an instance on the cloud? Would I still get charged by AWS if I just stop an instance? Any clarification in this area would also be much appreciated. Again, time is money!
>> I hope this helps many of us!
>>
>> Unfortunatly, I will not be in Pitt to ask these questions in person.
>>
>> Thanks in advance!!!
>>
>> Mike
>>
>> --- On Tue, 4/5/11, Lali <
laurafe@gmail.com> wrote:
>>
>> From: Lali <
laurafe@gmail.com>
>> Subject: Re: [galaxy-user] Analyzing Targeted Resequencing data with Galaxy
>> To: "Anton Nekrutenko" <
anton@bx.psu.edu>
>> Cc: "galaxy-user" <
galaxy-user@lists.bx.psu.edu>
>> Date: Tuesday, April 5, 2011, 11:50 AM
>>
>> Ohh sorry about that!
>> I am using both Windows XP and Ubuntu and I usually use Google Chrome.
>>
>>
>> On Tue, Apr 5, 2011 at 5:33 PM, Anton Nekrutenko <
anton@bx.psu.edu> wrote:
>> Lali:
>>
>> Please, always CC mailing list when you reply.
>>
>>> My only problem with Galaxy is that I have to keep on clearing my cache in order to get the history to display correctly, is there another way of solving this issue?
>>
>> Which browser/OS are your using?
>>
>> Thanks,
>>
>> anton
>> galaxy team
>>
>> On Apr 5, 2011, at 11:25 AM, Lali wrote:
>>
>>> Thanks so much for the tips Anton!
>>> I am very excited about the newer developments.
>>> I did watch the quickies and they were very useful for a beginner like me, I actually did my first try at the alignment by following the Illumina single-end tutorial video step by step, but you need to watch the paired-end too, for some of the first steps, which are explained better on that one.
>>> I have been playing around a lot with Galaxy, and I have several workflows, my department just started doing sequencing, so we don't have standard procedures set in place. I was assigned to evaluate Galaxy and CLC, and so far CLC has not impressed me, except for the fact that it can generate reports easily.
>>> I think Galaxy is the way to go for me (us, if I can convince them to run a local server), since I am not a bioinformatician, and just the fact that you can queue up actions and just walk away is fantastic (amongst other things).
>>> But because I am a beginner, I am not 100% of the settings I have chosen and my data is not looking too good so far, but I am having a bioinformatician come over and help me on Thursday and I think your tips will be of help.
>>> My only problem with Galaxy is that I have to keep on clearing my cache in order to get the history to display correctly, is there another way of solving this issue?
>>>
>>> Best regards,
>>>
>>> L
>>>
>>> On Tue, Apr 5, 2011 at 3:56 PM, Anton Nekrutenko <
anton@bx.psu.edu> wrote:
>>> Lali:
>>>
>>> In your case the workflow for capture re-sequencing should look like this:
>>>
>>> 1. QC data (groom fastq files and plot quality distribution)
>>> 2. Map the reads (use bwa)
>>> 3. Generate and filter pileup
>>> 4. Intersect pileup with coordinates of sure select bates.
>>>
>>> However, before you dive in please understand basic Galaxy functionality by taking a look at
http://usegalaxy.org/galaxy101 and watching *all* Illumina-related Galaxy quickies (black boxes on the front page on Galaxy). Next, take a look at
http://usegalaxy.org/heteroplasmy.
>>>
>>> Note, that we are working on bringing "industrial-strength" diploid genotyping functionality in Galaxy in the next two-three months that will include more sophisticated genotypers, recalibration and realignment tools, and novel visualization approaches.
>>>
>>> Thank for using Galaxy.
>>>
>>> anton
>>> galaxy team
>>>
>>>
>>>
>>> On Apr 5, 2011, at 2:44 AM, Lali wrote:
>>>
>>> > Hi!
>>> > I am having problems with my sequencing results, but I am a newbie at this; so I am thinking there is something wrong with my analysis. So far, I've tried Galaxy and CLC Workbench, but with CLC I could not align to the whole genome, only to individual chromosomes (maybe there is a way, but by the time the trial ended I had not found it).
>>> >
>>> > I used SureSelect capture kit and did single end sequencing on an Illumina. The files the lab sent me are FastQ Illumina 1.5 files, my samples were indexed, and I got a series of files each representing an Index.
>>> >
>>> > What would be the standard workflow for this kind of data?
>>> > Which tools/settings?
>>> >
>>> > Does anyone have an example Galaxy workflow for preparing (clipping adapters, quality trimming) and mapping Targeted Resequencing Data?
>>> >
>>> > Is there a way to obtain a coverage report through Galaxy?
>>> >
>>> > Is it possible to ignore/discard the reads mapped when the coverage is below a certain threshold?
>>> >
>>> > I know, I know, a lot of things, but I am very lost.
>>> > Any help is appreciated.
>>> >
>>> > L ___________________________________________________________
>>> > 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/
>>>
>>> Anton Nekrutenko
>>>
http://nekrut.bx.psu.edu
>>>
http://usegalaxy.org
>>>
>>>
>>>
>>>
>>
>> Anton Nekrutenko
>>
http://nekrut.bx.psu.edu
>>
http://usegalaxy.org
>>
>>
>>
>>
>>
>> -----Inline Attachment Follows-----
>>
>> ___________________________________________________________
>> 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/
>
> Anton Nekrutenko
>
http://nekrut.bx.psu.edu
>
http://usegalaxy.org
>
>
>
Anton Nekrutenko
http://nekrut.bx.psu.edu
http://usegalaxy.org