SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
Tuxedo suite not giving alternative TSSs their own loci Gordo2B Bioinformatics 0 07-07-2014 05:42 AM
GPU vs Phi: advice needed yaximik Bioinformatics 4 09-11-2013 11:11 AM
RNA-Seq: tuxedo suite transcription start site identification zillerm Bioinformatics 2 07-05-2013 09:58 PM
Software advice needed regarding SNP calling Burger Bioinformatics 3 05-18-2012 06:24 AM
ChIP-seq advice needed bcm Illumina/Solexa 2 03-22-2010 05:22 AM

Reply
 
Thread Tools
Old 08-12-2014, 01:10 PM   #1
thickrick99
Member
 
Location: Washington

Join Date: Jul 2014
Posts: 21
Post Advice Needed on Tuxedo Suite

Hi Everyone,

I am working on a project with two groups (A & B) each containing 5 samples. Within each sample I have two RNA-seq FASTQ reads which I downloaded. So as of now, I am planning to run tophat for each of the samples (for a total of 10 accepted_hits.bam files). From here I plan on running cufflinks for each sample with its respective .bam file to produce a total of 10 transcripts.gtf files.

My goal is to compare the differential gene expression of group A and group B. I am using the hg19 reference genome.

I have two questions. First, is there a way I can run tophat/cufflinks on all the samples all at once or do I have to run one sample, then wait until its done, then do the next sample and so on?

Second, once I have the transcripts.gtf files for each of the samples for groups A & B, is it necessary to merge them into one single .gtf file? I assume this would be done with cuffmerge right? What exactly does cuffmerge do and what are the advantages in using it. In addition, is there a need to use cuffcompare since I am comparing the differential gene expression of group A and group B or can I go straight to cuffdiff.

In general, my second question is essentially asking the pipeline for using software after tophat & cufflinks based on what I am trying to do. It would be great if someone could give me a suggestion as to the order of how I should approach this.

THANKS!
thickrick99 is offline   Reply With Quote
Old 08-12-2014, 04:23 PM   #2
dpryan
Devon Ryan
 
Location: Freiburg, Germany

Join Date: Jul 2011
Posts: 3,480
Default

If you have a cluster or single computer with sufficient RAM and cores then you can run as many instances of tophat/cufflinks simultaneously as you want.

If you don't use the -G option, then not using cuffmerge would cause you to compare apples and oranges (suppose one sample had a modified gene model and another didn't, the resulting FPKM comparison would be meaningless). If you use cuffmerge then you don't need to use cuffcompare. I think they had a methods paper with a step-by-step walk-through of an analysis, which I'd recommend you read through.
dpryan is offline   Reply With Quote
Old 08-13-2014, 10:22 AM   #3
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,170
Default

Is part of your objective to identify transcripts which have not yet been annotated in the human genome? If not then there is really no reason to use Cufflinks at all.

To perform DE analysis between groups A & B considering only already annotated genes in hg19 I would:

1. Download the hg19 reference set from the Illumina iGenomes site.

2. Prepare the known genes bowtie index as described in the TopHat manual.

3. Align your reads to hg19 with Tophat 2.x.x using the "-T" option (align to transcriptome only).

4. Count the reads aligned to genes from each of your 10 accepted_hits.bam (e.g. with feature counts or htseq-count).

5. Use the raw read counts matrix as input to your preferred DE module (e.g. EdgeR, DESeq2).
kmcarr is offline   Reply With Quote
Reply

Tags
cuff links, cuffcompare, rna-seq, tuxedo

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 01:18 PM.


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