Dear Noa and Bomba
i am also in situation like you, hardly any programming experiance but
want to analyse, bacterial tranmscriptome before and after an stress.
I am very new to galaxy, would be obliged if any one can give me more
hints, i have few queries
In Brief; we did our sequnecing by Hiseq 2000, from our partner i
got two files (FASTQ) for each treatment, for example R1 and R2
i was sucessful to upload these files (Fastq) and reference genome
file (Fasta) via FTP,
after upload, i run the Fastq groomer, with my understanding i must
quality filter my sequences, but i am not sure what shoud be the
cutoff.
after reading Noa email, seems like i should do Bowtie mapping, after
groomer , and filter is is there any other middle step also before i
go to bowtie
Another question is, should i join both the files (fastq joiner) at
any stage of analysis
Bomba could you please send me link as you stated, for RNA seqq
analysis on Univerity of albama website, although it is for eukaryote,
but may be will help me to understand the steps
Thanking you all
________________________________
To: Jeremy Goecks <jeremy.goecks@emory.edu>
Cc: "galaxy-user@lists.bx.psu.edu (galaxy-user@lists.bx.psu.edu)"
<galaxy-user@lists.bx.psu.edu>; closeticket@galaxyproject.org; Bomba
Dam <bomba.dam@mpi-marburg.mpg.de>
Subject: Re: [galaxy-user] Using galaxy for Bacterial RNA-seq
Hi Bomba
I have been recently struggling with the same issue (RNA-Seq on a
bacteria; no programming experience).
I was in touch with the authors of the tophat-cufflinks suite and they
all suggested that given that bacteria have little or no introns, you
can do bowtie followed by cufflinks and skip the tophat.
Alternatively, if you do decide to do tophat and then cufflinks, make
SURE to change the intron size parameters, otherwise you will get a
mess. I used min intron distance 10bp, max size 1000bp. If you just
want to do comparative work you can do bowtie and then cuffdiff
directly. Look at your data with the Galaxy genome browser after you
run tophat or bowtie to make sure it looks OK. (I found it easier to
convert bowtie data from SAM to BAM file and then to view it). One
thing I havent fully looked into is what happens at the ends of the
mapping (since bowtie assumes linear chromosomes and not circularized,
so just be aware of that difference as not all reads will align
properly at the ends).
Feel free to contact me if you need more help - I am definitely not an
expert but have been struggling through doing RNA-Seq on galaxy for
the past month or so, so may be able to help with some things. Make
sure you use matching gtf reference annotation (if you have this) and
genome!
Good luck!
noa
Bomba, I'm not familiar enough with bacterial/prokaryotic
transcriptomes to suggest a possible workflow. You might try the
standard Tophat-Cufflinks-Cuffcompare/merge-Cuffdiff workflow and see
whether you get meaningful results; Tophat runs Bowtie internally, so
there's no reason to run Bowtie separately unless there are Bowtie-
specific parameters that you need to modify. I've had very little
experience with PALMapper and can't speak to its efficacy, either for
eukaryotic or prokaryotic transcriptome analyses. Finally, I've cc'd
the galaxy-user mailing list. Using this list is the best way to reach
the Galaxy user community and get in touch with someone that has used
Galaxy to analyze bacterial transcriptomes. Good luck,
Most examples provided and used for analysis are from eukaryotic
systems. I am a bit confused weather the same workflow will also work
well for bacterial systems as there are no splicing events or I should
make some modifications. Can you kindly suggest me which workflow
should I follow for mapping the bacterial reads (Bowtie, Tophat or
PALMapper) and subsequent quantification steps. I want some guidance
in this regard. With kind regards, Bomba Dam
--
Dr. BOMBA DAM
Alexander von Humboldt Postdoctoral Research Fellow
Max-Planck-Institut für terrestrische Mikrobiologie
Karl-von-Frisch-Straße 10
D-35043 Marburg, Germany
E mail: bomba.dam@mpi-marburg.mpg.de PHONE: +49 176 321 321 75
(Mobile); +49 6421 178 721 (LAB); +49 6421 2828516 (ROOM) Assistant
Professor of Microbiology
Department of Botany, Institute of Science
Visva-Bharati (A Central University)
Santiniketan, West Bengal 731235, India.
E mail: bumba_micro@visva-bhatari.ac.in, bumba_micro@rediffmail.com;
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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
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