metatranscriptomics protocol

[furor]

Hi all,


we're planning to do some metagenomics and metatranscriptomics runs of 5 sediment samples along a depth gradient in a lake, to observe shifts in functioning/expressing and composition with changes in a.o. light and oxygen conditions.

We'd run our libraries on an Illumina HiSeq 2500.

As we don't really have any experiences with this kind of sample preparations (RNA), I've composed a protocol.
The main goal is to target mRNA, but perhaps small RNAs might be interesting too. We want to target the complete community, i.e. eukaryotes, prokaryotes and if possible viruses.
The lake is oligotrophic and a relatively low diversity is present (low complexity of food web, mostly unicellular and few small multicellular eukaryotes)

Could you please comment on the following protocol? Are there better options? Suggestions?

Samples are already taken. LifGuard solution was added for transport (2 months!) and samples were stored at -80°C (also during transport).

Lyophilization of total RNA for TruSeq is not really possible and additionally we don't want to take the risk of possible degradation.

- Extraction:

*MoBio Powersoil RNA extraction kit
(compatible with the Lifeguard solution; removes humic acids which might be present)
*MoBio RNA PowerSoil® DNA Elution Accessory Kit
(we do have separate neighboring cores for metagenomics, but this might be a more sound approach)

- Removal of possible cary-over DNA
Turbo DNA-free
(DNAse is removed with the kit, but is an additional phenol/chloroform step wanted?)

- rRNA depletion
RiboZero Gold epidemiology kit

- cleaning
RNeasy MinElute Cleanup Kit

- cDNA synthesis
Universal RiboClone cDNA Synthesis System

- library prep
Nextera


Are there better alternatives? I'm not very sure abou the cDNA synthesis.
Am I oblivious to some issues?
General hints?

While diversity is relatively low, some depths are dominated by mosses. Does anybody know if it is possible to separate the moss fraction from the rest, as we fear that moss RNA/DNA might engulf the rest?
I'm thinking of first vortexing the samples, pour them over a filter/microsieve, and consequently flush to recover as many epibionts as possible. Or would I jeopardise the RNA integrity?

Btw, we have 5 subsamples/sample of which we would sequence 3 unpooled. One additional subsample would not have the rRNA depleted and also sequenced (but not as deep), so we would have an idea of who is active, without the (additional) bias of the Ribozero kit (as not all rRNA is removed).
Would this be useful?

Thanks in advance!
Kind regards.

[kerplunk412]

Is there any reason you want to do the cDNA and library prep separately? Otherwise it seems like going into library prep after rRNA depletion using a directional RNA-Seq kit would be a much better option. I don't think a cDNA/Nextera protocol will allow you to retain strandedness.

Otherwise here are a few comments:
-You won't be able to recover many small RNA reads with a typical RNA-seq protocol, but you should be able to recover lncRNA, which is another good reason to use a directional protocol. Small RNA libraries are a whole other beast.
-I would not think an additional DNA removal step would be needed if a DNAse treatment is included in the RNA extraction protocol, but this should be fairly easy to test. Any additional steps reduce yield and increase chance for contamination, degradation, or error, so this is something to consider.
-If possible, use the maximum RNA input recommended by the manufacturer of the protocol you end up using. This is always good practice, but especially useful in rRNA depleted samples because the residual tRNA (mostly) causes many people to think rRNA depleted samples have more mRNA than they actually do.

If you would like some suggestions on RNA-Seq kits that would be appropriate for your project please feel free to PM me.

[furor]

Hey, tried to pm you but doesn't work. Directional kits might be interesting!

[cpavloud]

Hi!

You could use the TruSeq Stranded mRNA Library Prep Kit for your samples.
It is also something I am thinking of doing with my sediment samples, at a MiSeq.

According to the protocol, you can start with 0.1 - 4 μg of total RNA and then select the mRNA with the poly-T oligo beads, prepare the cDNA and sequence.

It seems pretty straightforward which probably means it isn't...
I don't know if anyone else has tried this kit on environmental samples... Some input would be highly appreciated.

[vivi-Jasmine]

Hi furor,

I'm trying to do metatranscriptomic analysis these days. I'm wondering do you have any suggestion for me? Is it necessary to do mRNA enrichment? Which protocol you choose to perform cDNA synthesis and library pre?

Thanks a lot.
All the best.

[adam.geber]

vivi-Jasmine, what kinds of samples are your working with and for what purpose? That will determine your needs for enrichment and whatever follows.

[vivi-Jasmine]

vivi-Jasmine, what kinds of samples are your working with and for what purpose? That will determine your needs for enrichment and whatever follows.

Dear Adam,

Thanks for you reply. I'm working on rhizosphere soil sample. We want to do metatranscriptomics of microbial community. Have you have any suggestion on this?

[adam.geber]

Dear Adam,

Thanks for you reply. I'm working on rhizosphere soil sample. We want to do metatranscriptomics of microbial community. Have you have any suggestion on this?

Enriching for your transcripts of interest can only be helpful! What are your RNA yields like and how intact do they appear to be? Do you have any sense of the composition of your community (i.e. bacteria, archaea, fungi, protists) and do you expect there to be any plant tissue present? Characterizing the integrity of RNA from a noncanonical or complex microbial community can be quite difficult but if you can assess your sample on a Bioanalyzer/Tapestation that should give you a good approximation. Can I assume you want to use an Illumina platform, by the way?

If you're only interested in the bacterial community you could use either the Ambion MICROBExpress mRNA Enrichment Kit or Illumina's Ribo-Zero Gold rRNA Removal Kit. The input requirements are lower (500ng) for the latter option but I believe the price is considerably higher. Alternatively, NuGen's Ovation Complete Prokaryotic RNA-Seq system might be a good option if you have lower yields (>100ng) -- this method removes rRNA at the level of library prep by selectively removing sequencing adaptors from cDNA inserts.

[vivi-Jasmine]

cDNA synthesis

Hi Adam,

I'm only interested in bacterial and archaea community and I will try to extract RNA tomorrow. So i'm not sure about the quality and yield now. After I get the result. I will try to do the next step as you suggested. I'm wondering which cDNA synthesis kit are you using? thanks again.

Ling

Enriching for your transcripts of interest can only be helpful! What are your RNA yields like and how intact do they appear to be? Do you have any sense of the composition of your community (i.e. bacteria, archaea, fungi, protists) and do you expect there to be any plant tissue present? Characterizing the integrity of RNA from a noncanonical or complex microbial community can be quite difficult but if you can assess your sample on a Bioanalyzer/Tapestation that should give you a good approximation. Can I assume you want to use an Illumina platform, by the way?

If you're only interested in the bacterial community you could use either the Ambion MICROBExpress mRNA Enrichment Kit or Illumina's Ribo-Zero Gold rRNA Removal Kit. The input requirements are lower (500ng) for the latter option but I believe the price is considerably higher. Alternatively, NuGen's Ovation Complete Prokaryotic RNA-Seq system might be a good option if you have lower yields (>100ng) -- this method removes rRNA at the level of library prep by selectively removing sequencing adaptors from cDNA inserts.