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 <laur...@gmail.com> wrote:


From: Lali <laur...@gmail.com>
Subject: Re: [galaxy-user] Analyzing Targeted Resequencing data with Galaxy
To: "Anton Nekrutenko" <an...@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 <an...@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 <an...@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/
___________________________________________________________
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/

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