Gene Expression through RPKM

[pfranchini]

Hi all,
I am a new user of the next generation sequencing.
I am working with Illumina output sequence reads of a non-model organism so I have not a reference genome. I would like to quantify gene expression of diffrent group of individuals through the CLC Workbench (mRNA sequencing tool based on the RPKM concept). Do you think can I use the the assembled contigs as a reference genome and use this tool to compare gene expression in different individuals?
Thanks!!!
P

[Patrick Dekker]

Hi all,
I would like to quantify gene expression of diffrent group of individuals through the CLC Workbench (mRNA sequencing tool based on the RPKM concept). Do you think can I use the the assembled contigs as a reference genome and use this tool to compare gene expression in different individuals?
Thanks!!!
P

Hi,

Yes, you can use assembled contigs as a reference genome.
First run a de novo assembly of all the reads of all individuals. Use the output of this assembly as reference for the mRNA seq.

Patrick Dekker,
(I'm the developer of this mRNA-seq module )

[caroline]

NaN in mRNA-Seq

Can you tell me what NaN stands for in the RNA-Seq expression value analysis within CLC?

[Patrick Dekker]

RPKM can only be calculated when an gene has an mRNA annotation. (RPKM is number of reads which map per kilobase of exon model per million mapped reads).

For genes without an mRNA annotation we can't calculate RPKM because we don't know the length of exon model. So for these genes we report NaN (Not an Number). If you want to do some statistics for these genes, you can use an other column as expression value

Patrick

[caroline]

NaN Followup

In your opinion is RPKM the best overall output option to use for RNA-seq?