Hi Elsayed

At the risk of overwhelming you with examples:
The Genomics Virtual Lab also provides basic and advanced RNA-seq and Variant 
Detection tutorials; with a (small) public Galaxy instance specifically aimed 
at running these two tutorials.
https://genome.edu.au/wiki/Learn

Ron Horst
Project Manager, Genomics Virtual Lab
Level 6, QBP, The University of Queensland, QLD 4072
t.  07 3346 2276 |  m.  0417  538 723 |  e. r.ho...@uq.edu.au | w. 
www.genome.edu.au<http://www.genome.edu.au/>



From: galaxy-dev-boun...@lists.bx.psu.edu 
[mailto:galaxy-dev-boun...@lists.bx.psu.edu] On Behalf Of Jennifer Jackson
Sent: Saturday, 4 January 2014 6:59 AM
To: Elsayed Hejazy
Cc: Galaxy Dev
Subject: Re: [galaxy-dev] Help Needed

Hello Elsayed,

Your protocol below seems to be a mix of a variant detection and an RNA-seq 
workflow. To build a workflow for RNA-seq, you will want to compare your steps 
with the protocols in the link that I sent you.

If you want more examples, many more can be found here (for both RNA-seq and 
variant analysis, these are distinct analysis). Some have workflows that you 
can import and use directly or modify.

   https://wiki.galaxyproject.org/Learn#Other_Tutorials

   Shared Data -> Published Pages

In particular, there is a sample workflow in this tutorial that will help you 
to understand what the different steps are for and what order they go in.
https://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise

Best,

Jen
Galaxy team


On 1/3/14 10:57 AM, Elsayed Hejazy wrote:
Dear Dr. Jennifer,
A lot of thanks for your reply its really mean alot for me.
i am still NGS junior i trying to do the following steps kindly give me the 
right order for these procedures
Data Description: i have to samples each sample consists of 14 FASTQ file (7 
forward and 7 reverse ) i think this mean its paired end from Illumina then i 
will try the following workflow to got best results
1- Drag tow input dataset into workflow one for forward sequences file and one 
for reverse to use paired end option in TOPHAT tool later and when i run this 
workflow i will select multiple selection for the 7 forward files to analyse 
all of them at the same time
2- Drag FASTQC and link with last step for each to got if these file may be 
illmina 1.8 version or older.
3- Drag FASTQ Groomer and link with last step if files older than 1.8 version 
to prepare as .FASTQSANGER format.
4- Drag Filter FASTQ and link with last step to remove redundancy of sequences.
5- Drag FASTQ trimmer to remove unwanted ends of sequenced may occur
6- Drag Manipulate FASTQ and link with last step (i dont know why).
the above six steps done twice to generate to files as output to make as input 
for the following steps.
7- Drag TOPHAT for illumina and make it accept paired end files and link each 
file generated from QC to TOPHAT this step used to align and map with reference 
genome.
8- Drag Cufflinks and link with aligned BAM file generated from TOPHAT to 
create an assembly
9- Drag Cuffmerge and link with GTF file from Cufflinks this step to merge all 
assemblies generated in Cufflinks
10- Drag Cuffcompare and link with last step to got detailed reports for 
accuracy of all generated assemblies.
11- Drag MPileup and link with TOPHAT BAM file to generate file containing SNPs 
sites.
12- Drag Pileup-to-Interval and link with MPileup step to filter the number of 
output SNPs to successive one or the most accurate. (i dont know what is the 
difference between this tool and Filter Pileup).
- i dont know what is the tools used to know the copy number variation CNV
i need to know how to separate human sequences from sample may infected with 
any other sequences (is this at alignment stage)
i need to know the perfect order of steps if this order is not completely right.

Is this what i should do to make a good NGS workflow got all possible 
information form dataset

Really i am so so so sorry for disturbance - waiting your reply
Best Regards,
elsayed


On Thu, Jan 2, 2014 at 6:05 PM, Jennifer Jackson 
<j...@bx.psu.edu<mailto:j...@bx.psu.edu>> wrote:
Hello Elsayed,

Protocol help for RNA-seq analysis can be found here:
https://wiki.galaxyproject.org/Support#Tools_on_the_Main_server:_RNA-seq

The QA/QC steps should be done before mapping, on individual datasets (such as 
replicates) or on partial or merged datasets as needed (if that is how the data 
was sequenced, just be consistent). Only keep in mind that the larger the 
dataset, the more compute some of these steps can require.

Hopefully this helps,

Jen
Galaxy team

On 1/1/14 1:32 AM, Elsayed Hejazy wrote:
i need to know more about the order of steps for RNA Seq data analysis
Is alignment and assembly should done first to combine all FASTQ reads into one 
file and start analysis or i should do Quality Control ,filtering  , trimming 
and manipulation first and do alignment and assembly at the end of analysis to 
use tophat for example or any other analysis tools in Galaxy

Thank you
elsayed



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Jennifer Hillman-Jackson

http://galaxyproject.org




--

Jennifer Hillman-Jackson

http://galaxyproject.org
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