How to analyze RNA-seq data ?

bioR · 11-03-2010, 11:11 AM

Hii all,
I am new to Bioinformatics field, i have never worked on any kind of statistical data analysis using tophat or bowtie or cufflink, so please excuse me for my naiveté. My querry is as follows:
I have two samples to compare one is a control and other is a knock-in. I did their RNA extraction -> cDNA prep -> Library prep -> Sequencing on Illumina Hi seq -> tophat -> bowtie -> cufflink -> Cuffcompare -> Cuffdiff.
Now after these many analysis i have so many output file (.gtf, .tracking, . refmap, .expr and so on) and i am so much confused as how and what to look into them.
Can anybody please tell me of any tutorial available where is can have an idea of how to further analyze my data in a step by step manner and find out significant differences between both of my samples.

Thanks in advance.

malachig · 11-04-2010, 08:38 PM

Hello BioR,

Welcome to the field of bioinformatics! Our own member 'MDY' wrote such a tutorial here.

My next piece of advice is to keep digging through those files and try different tools. You will learn a lot experimenting and you may find tools that more directly give you the answers you seek.

There are many such tools mentioned in this forum, but I will recommend 'ALEXA-seq' since that is one I had a hand in creating. Although more complicated to run, and less elegant in implementation, the final result is in my opinion more intuitive and interpretable than Cufflinks. Keep reading the forum for more ideas on tools to use.

Finally, tell us more about the experimental details and what you hope to get out of the data. Straight gene expression? Novel transcripts? Gene fusions? Alternative splicing? Mutations? Allele specific expression? RNA editing? Etc...

RNA-seq data has many possible uses and each has its own analytical approaches...

sstarkenburg · 02-09-2011, 10:51 AM

Does Alexa-Seq only work with paired end data or does it support single end Illumina reads as well? Second, I will be using a "novel" assembly from an algae that is currently not annotated. Can Alexa-Seq handle this?

Mokinhas · 09-12-2013, 08:08 AM

Dear all,
I am quite new to bioinformatics and R enviroment. Nevertheless Im trying my best to analyse data from RNA seq. I ve done everything to get CummerBund funcioning and that went not smooth but it is working good now

Neverheless when I want to plot the data I get the following error:

fpkmSCVPlot(genes(cuff))
Scale for 'x' is already present. Adding another scale for 'x', which will replace the existing scale.
Error in seq.default(from = best$lmin, to = best$lmax, by = best$lstep) :
'from' must be of length 1
In addition: Warning message:
In max(log10(dat$fpkm)) : no non-missing arguments to max; returning -Inf

Can please please please help me to understand what this is?
Much appreciated,
xxx

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11-03-2010, 11:11 AM	#1
bioR Junior Member Location: USA Join Date: Nov 2010 Posts: 1	How to analyze RNA-seq data ? Hii all, I am new to Bioinformatics field, i have never worked on any kind of statistical data analysis using tophat or bowtie or cufflink, so please excuse me for my naiveté. My querry is as follows: I have two samples to compare one is a control and other is a knock-in. I did their RNA extraction -> cDNA prep -> Library prep -> Sequencing on Illumina Hi seq -> tophat -> bowtie -> cufflink -> Cuffcompare -> Cuffdiff. Now after these many analysis i have so many output file (.gtf, .tracking, . refmap, .expr and so on) and i am so much confused as how and what to look into them. Can anybody please tell me of any tutorial available where is can have an idea of how to further analyze my data in a step by step manner and find out significant differences between both of my samples. Thanks in advance.

09-12-2013, 08:08 AM	#4
Mokinhas Junior Member Location: Amsterdam Join Date: Sep 2013 Posts: 4	RNA seq CummerBund error Dear all, I am quite new to bioinformatics and R enviroment. Nevertheless Im trying my best to analyse data from RNA seq. I ve done everything to get CummerBund funcioning and that went not smooth but it is working good now Neverheless when I want to plot the data I get the following error: fpkmSCVPlot(genes(cuff)) Scale for 'x' is already present. Adding another scale for 'x', which will replace the existing scale. Error in seq.default(from = best$lmin, to = best$lmax, by = best$lstep) : 'from' must be of length 1 In addition: Warning message: In max(log10(dat$fpkm)) : no non-missing arguments to max; returning -Inf Can please please please help me to understand what this is? Much appreciated, xxx

11-04-2010, 08:38 PM	#2
malachig Senior Member Location: WashU Join Date: Aug 2010 Posts: 117	Hello BioR, Welcome to the field of bioinformatics! Our own member 'MDY' wrote such a tutorial here. My next piece of advice is to keep digging through those files and try different tools. You will learn a lot experimenting and you may find tools that more directly give you the answers you seek. There are many such tools mentioned in this forum, but I will recommend 'ALEXA-seq' since that is one I had a hand in creating. Although more complicated to run, and less elegant in implementation, the final result is in my opinion more intuitive and interpretable than Cufflinks. Keep reading the forum for more ideas on tools to use. Finally, tell us more about the experimental details and what you hope to get out of the data. Straight gene expression? Novel transcripts? Gene fusions? Alternative splicing? Mutations? Allele specific expression? RNA editing? Etc... RNA-seq data has many possible uses and each has its own analytical approaches...

02-09-2011, 10:51 AM	#3
sstarkenburg Junior Member Location: New Mexico Join Date: Aug 2010 Posts: 5	Does Alexa-Seq only work with paired end data or does it support single end Illumina reads as well? Second, I will be using a "novel" assembly from an algae that is currently not annotated. Can Alexa-Seq handle this?