Question: RNA-Seq of environmental samples
0
gravatar for Biogeek
3.2 years ago by
Biogeek350
Biogeek350 wrote:

Dear all,

I want to ask, has anyone had any experience with carrying out RNA-Seq on environmental samples? If so, did you ever find transcripts from other organisms which are expected to live in the same environment in your blast results.

Are there any methods or publications out there which have a protocol to follow for dealing with possible contamination and possibly the removal of such transcripts from the data?

Thanks.

ADD COMMENTlink modified 3.2 years ago • written 3.2 years ago by Biogeek350

Genuinely curious, what's an environmental sample?

ADD REPLYlink written 3.2 years ago by jotan1.2k

A sample from the field . Just aware many people on here work on biomedical and cell lines which Prob do not have much contamination.

ADD REPLYlink written 3.2 years ago by Biogeek350

Animal, vegetable or bacterial? Do you isolate cells first? Is there a reference genome?

If you're mapping to a large reference, the contaminants should be mostly eliminated at this step. You could also map the reads onto multiple organisms (target + potential contaminants) and discard any reads which map to the contaminants. Tedious and you might potentially lose informative reads but you'll have to decide whether you'd prefer sensitivity or selectivity.

ADD REPLYlink written 3.2 years ago by jotan1.2k

Unless you are planning to collect samples under non-sterile conditions/transport or store without controlling temperature/allow direct contact with collector's hands etc why would there be a chance of contamination?
If you are planning to isolate RNA without culturing then you will have no idea what you are going to get. It would depend on the method you use for RNA isolation and/or library prep done afterwards.

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by genomax69k

Genomax2, Assume your organism is vegatative and you have opportunistic species growing on it within the environment. Some organisms are not quite up to speed with culturing/ cell lines etc, so the only option is to take your sample from the field and clean it as well as you can without being harsh. If you do deep sequencing it is logical you will pick up transcripts from possible contaminating organisms (of those which aren't the target organism). This is the scenario I am talking about. Now if you enrich the mRNA, you will stick get contaminants from eukaryotes... I guess Blast is a way to filter, would people agree?

ADD REPLYlink written 3.2 years ago by Biogeek350

Don't blast and filter, just embrace the metagenome you're sequencing. The contaminants are like lower-expression genes. Hope theyre under 50% of the sample.

You can align the reads to each genome you have or make one big collection of chromosomes and work with that.

ADD REPLYlink modified 3.2 years ago • written 3.2 years ago by karl.stamm3.5k

Hmmm, but it's not technically a meta-genome. It is a de novo transcriptome. If a reference existed, it would make life easier ;-) I'd assume the contaminants are lower expression, I will do some exploration of the data and see what these transcripts represent in counts. Yeh, well under 50%, talking about 1/2%.

ADD REPLYlink written 3.2 years ago by Biogeek350

What you are describing here is different than what many would consider "environmental" sampling (which would normally mean you collect a water sample, filter a sample of air or collect a sample from soil etc).
It sounds like you have a certain organism (that you can selectively collect albeit from a natural location) that may have other living things associated with it. As you said, you would have to do your best to clean the specimen and do the rest of the prep. You would be able to filter obvious reads that don't belong out (blast could be one option). Is there a reference or a related genome available? That can make this read decontamination simpler.

ADD REPLYlink written 3.2 years ago by genomax69k
1
gravatar for WouterDeCoster
3.2 years ago by
Belgium
WouterDeCoster40k wrote:

I would suggest to start with DNA sequencing of your sample to get an idea what's in there, perhaps 16S?

ADD COMMENTlink written 3.2 years ago by WouterDeCoster40k
0
gravatar for Biogeek
3.2 years ago by
Biogeek350
Biogeek350 wrote:

decosterwouter, is 16S not primarily for prokaryotes. If anything 18S, but if you enrich for mRNA instead of ribosomal RNA, how do you determine what's in your RNA-Seq sample.I'm still assuming blast is the best bet. I've noticed a new tool called decon, but I think it's more orientated towards genome and meta-genomics rather than transcriptome.

ADD COMMENTlink written 3.2 years ago by Biogeek350

I'm working on human genetics so excuse my ignorance about metagenomics and identification of species ;-)

ADD REPLYlink written 3.2 years ago by WouterDeCoster40k
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