RNA_seq on (non-model) less well annotated genomes

[Drerio]

Hi All -

I am new to the forum, but I am hoping that you might be able too help me parse a question of library preparation. I am about to embark on an RNA seq project on an Illumina HiSeq2000 to compare behavior responsive transcripts in the brains of three insect species, all of which should have annotated genomes by the time we have data back, but the genomes aren't out yet. It is going to take a lot of libraries from brains (200+) to narrow down the transcripts specific to the behavior we are interested in, which means this is going to be a very expensive and time consuming project. Right now, we are trying to figure out which protocol to use to make our libraries. The options we have considered are 1) TruSeq, 2) ScriptSeq v2, and 3) the updated protocol from the recently published Wang et al paper. At this point, each method has its advantages: TruSeq is well described and well worked out, 2) ScriptSeq is strand-specific and supposedly takes about half the time to make each library, and 3) the Wang protocol looks pretty inexpensive and is again strand-specific. As a side note, we have been interested in the strand-specificity because we have thought that it might help us describe our genomes better, giving us better quality data for cross-species comparisons.

Has anyone thought through the strand-specific vs non-specific issue for annotating non-model systems? Is the state of the art using strand-specific protocols, or does it not really matter for most users? Does anyone have a strong opinion about any of these library preparation methods?

Thanks a lot for any help!

[Marianna85]

Hi Drerio,
we are going to start constructing libraries in-house and we are wondering the same questions as you. The situation is similar, with a non-model species and less annotated genome.
Have you decided yet?
Someone have news???
Thank you all

[Drerio]

We are still trying to figure it out as well. We may end up with strand-specific data whether we really wanted it or not as we are currently pursuing (as one option) the paper below.

Others have clearly assembled transcriptomes using non-strand-specific library preparation protocols, but it appears that some groups have found that they have an easier time with strand-specific versions if they are working with particularly compact genomes such as bacterial species. The other advantage of strand-specific protocols is that they should allow researchers to sort out anti-sense transcripts more easily than with conventional approaches. This might be useful if you have less information about how your transcriptome is assembled as well.

At the moment we have three concerns. First, we are still investigating software able to use the strand-specific information. We aren't quite sure how it will go building a transcriptome using it. Second, some reports suggest that strand-specific protocols may have bias at one end or the other of transcipts. Third, the more recent strand-specific protocols (such as the one below and ScriptSeq v2) haven't yet been widely compared to the most widely adopted method, TruSeq. On the last point, I have been in contact with a lab in Denmark which is about to adopt ScriptSeq v2 for a large project, and I have been in contact with the authors of the protocol below. Both groups assure me that their data look good, and they have or will be looking at hundreds of samples. One last point: if you do choose to pursue the protocol below, I recommend contacting the authors as they have modified the protocol since it was published.

Good luck!

A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq.

Wang L, Si Y, Dedow LK, Shao Y, Liu P, Brutnell TP.

PLoS One. 2011;6(10):e26426. Epub 2011 Oct 19. Erratum in: PLoS One. 2011;6(11). doi: 10.1371/annotation/e5ef7afc-7e81-4053-8670-1bb3402f63fd.

PMID:
22039485
[PubMed - indexed for MEDLINE]

[Marianna85]

We are considering all the three protocols you mentioned, and we are interested in strand specific protocol too, because we prefer to obtain reads which own a additional biological value.
I also found a protocol from NuGene, I'm studing it in these days... it seems to be good, especially if you start with a low quantity of total RNA. Did you find it?
It has few advantages comparing with other protocols: you can start with total RNA without mRNA enrichment or rRNA reduction (because of the cDNA synthesis technology), there isn't gel purification steps and it seems to be possible avoiding the fragmentation step with Covaris.
Unfortunately papers using this protocol have not been published yet.

[zhou junfei]

Could you give me this papers ? I have some trouble about the fragmenttaion of Riibominus RNA during the construction of total RNA-seq library. I have used the protocol suppled by covaris mRNA and total RNA fragmentation, but I couldn't found the size range in 100~200nt，even sheared 420s, eout of the standard 300s.

Please send to this email [email protected]，if possible.
Thanks a lot!

[Marianna85]

I don't understand what you need Zhou, which papers?