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Old 04-10-2014, 04:06 AM   #1
dodo1981
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Default Gene expression between different organisms

Hi everybody,

I need your help regarding the gene expression analysis.

I am working on genes from 9 Amoeba species.
My Proff. from ... said that next step of my analysis should be to do the gene expression of the genes I have. I did annotation before, assembly of transcriptome, and all analysis which should be done after sequencing. I am using the RNA-Seq data.

I thought the gene expression analysis could be done only within one species. Example I would have three the same genes "AB" but from different Yeast growth stage. I could check the gene expressions between them and find example: that on log stage the gene AB is overexpressed compared with the same gene but from the dying phase. Is that correct? Hope I explained simply.

I am working on 12 proteins (genes) but in 9 species of Amoeba. Some distance between these Amoebas is huge, some smaller.
How it is possible to do the gene expression of the 12 genes between these species?
Is that even possible?

I am sorry I just started to analyse what should be my next step of the PhD project, this is the reason if the question is "stupid" please take my appologize.

Thank you very much
Dorota
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Old 04-10-2014, 04:32 AM   #2
areyes
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Hi Dorota,

I had a similar problem trying to do comparative transcriptomics of exon usage regulation in primate species. I can give some input that I hope could be useful for you, but first let me ask a couple of questions to make sure I understand completely:

1. How exactly is your experimental design? If I understood correctly, you have RNA-seq data from 9 Amoeba species? Same tissues? several tissues? what conditions? do you have replicates (i.e. several individuals per species?)?

2. What is precisely the question you want to answer? Differences in expression between your Amoeba species? Do you have reference genomes for all the amoebas or you want to be based on your transcriptome assemblies?

Alejandro
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Old 04-10-2014, 05:28 AM   #3
dodo1981
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Hi Alejandro!
Thank you for the quick answer. I would be very happy if you could help me and give me some good feedback.

Now the answers:
1) First step of my Phd was to find (and annotate) 12 proteins of interest in 9 species of amoebas. I have RNA-Seq data for 2 of these 9 amoebas (own experiments). For these two amoeba species I have already assembled their transcriptomes (de novo using Trinity). Material for sequencing was collected in the same growth stage. For remaining 7, I don't know the exact sampling conditions, but the data (transcriptomes and genomes) are available in Genbank, so maybe information is there. One problem: there is no genome sequence for one of our two amoebas.
About replicates: I have two replicates (two rounds of sequencing) for one of the amoebas. They are from the same total RNA but sequenced by two different platforms: HiSeq and GA2.

2) I am interested in analyzing gene expression of the 12 proteins in all 9 species of Amoeba. Yes, differences in expression between the amoebas. As I wrote above: the genomes are available, with exception of one of the sequenced amoebas for which I have assembled transcriptome.

Thank you Alejandro and I am looking forwar to see your answer

Dorota
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Old 04-10-2014, 06:40 AM   #4
mbblack
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I think areyes was trying to get at two points you need to clarify.

1. Differential expression is just that, a purely relative measure of expression changes when compared to some arbitrary baseline. So you need a common baseline in order to compute differential expression across your 9 species for comparison. What, exactly, is your intended baseline? Is there an accepted common ancestral species you can use as a baseline for each of these, or a single most common species found in the same habitat? Bottom line is you need one common genome to use as the reference genome for the differential expression computation for the other 9 species you wish to compare.

E.G. In an experiment of some xenobiotic exposure to the these animals, your common baseline would be your untreated control samples. You would compute differential expression in all your treated groups relative to those untreated animals. In your case, since it sounds like you'd like to just compare innate levels of expression between species, you need some common denominator to use as a baseline - is one of these species clearly the most common of the 9 in the habitat(s) you collected from?

2. In order to have any statistical significance of differential expression, you must have actual biological replicates. Without them, you have no way to determine the inherent variance of expression within each species, and without knowing that variance, you simply cannot compute statistical significance of your sample relative to your baseline.

So, were these species collected as pooled samples of several individual amoeba each? If so, did you run multiple sets of pooled amoeba from each species (at least 3, preferable 5 or more in order to capture the range of inherent intra-species variability)?
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