SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
cuffdiff for differential gene testing Paul Walker RNA Sequencing 4 01-26-2015 08:04 AM
Does Cuffdiff normalize FPKMS before it does differential expression? greener Bioinformatics 0 02-25-2011 02:37 PM
Cufflinks/Cuffdiff significant differential expression memo Bioinformatics 5 01-25-2011 09:49 AM
Cuffdiff differential expression two groups jamminbeh Bioinformatics 0 12-09-2010 02:13 PM
Differential gene expression of gene clusters anjana.vr RNA Sequencing 1 10-28-2010 10:33 AM

Reply
 
Thread Tools
Old 05-10-2012, 12:59 AM   #1
kumardeep
Member
 
Location: Chandigarh, India

Join Date: Apr 2012
Posts: 15
Default Query regarding the cuffdiff command to get differential gene expression

Hi
I am using RNA-seq data to use "Differential gene expression". For that I have taken data from SRA (NCBI) raw files of human sample in some experiment (Control- 3 files; each of size ~1GB and test -3 files; size ~1GB).
(1) I downloaded corresponding *.sra files.
(2) After converting these (all 6 files) into fastq and filtering these reads, I mapped these reads using BOWTIE onto reference genome hg19.fa from UCSC browser.
Command I used is:
bowtie2 -S *.sam -x reference.build -U *.fastq_filtered
(3) Next used samtools to convert *.sam to *.bam files through this command :
samtools view -bS *.sam >*.bam
(4) Next command is :
samtools sort *.bam *.sorted
(5) 4th step gave me *.sorted.bam files for all 6 input files.
(6) Then I used cufflinks to proceed further to get RPKM values. command is
cufflinks *.sorted.bam
(7) Query is :
(a) Do I have to run the above command in 6th step for all 6 *.sorted.bam files (3 for control and 3 for test sample ) ?
(b) Also, How to use cuffdiff to finally get quantification of each gene with relative RPKM values ?
(c) As far as I understand, Gene names are not available in these files; so how to tag gene names with corresponding RPKM values (i.e. this RPKM values is for this gene or that gene) ?
(d) Also, How to say that a given gene is overexpressed or underexpressed in test sample as compared to control sample ?
(e) Also, How to average the replicates (as I have 3 control replicates and 3 test replicates) ? The cuffdiff command is not contemplated by me which says like this :

cuffdiff [options]* <transcripts.gtf> <sample1_replicate1.sam[,...,sample1_replicateM]> <sample2_replicate1.sam[,...,sample2_replicateM.sam]>... [sampleN.sam_replicate1.sam[,...,sample2_replicateM.sam]]

(f) Is it necessary to run cuffcompare command in this case ?

Thanks in advance
kumardeep
kumardeep is offline   Reply With Quote
Old 05-24-2012, 09:07 AM   #2
chknbio
Member
 
Location: Baltimore

Join Date: May 2012
Posts: 14
Default

I am also looking for an answer to (d) listed in your post. Did you find an answer to this question? Or does anyone have an answer to how to you determine, output the genes that are upregulated or downregulated compared to control or other samples in the experiment?

Thanks!
chknbio is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 12:55 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO