Hello, We have received some very interesting responses to our problem regarding RNA-Seq vs. Tag-Seq. Just in case someone might be interested, here are all responses. Thanks to everyone for all their help, Lucia Original question Hello all, My name is Lucia and I am a Biology PhD student from University of Buenos Aires, Argentina. For my thesis I am exploring the changes the bacteria Wolbachia pipientis induces in the weevil Pantomorus postfasciatus in order to make infected females parthenogenetic. We know there is a bacterial density threshold necessary to cause such a radical change in the weevil's reproductive system and one of my objectives is to identify differentially expressed genes in ovarian tissue from sexual females, infected females and "cured" females (females treated with antibiotics to reduce the bacterial density below the threshold) in order to shed some light on how Wolbachia affects the reproductive system of infected weevils. Two paths lay ahead of me: standard RNASeq or TagSeq. From what I have been reading, TagSeq is a low-cost but still reliable alternative (Meyer et. al., 2011 Vdoi: 10.1111/j.1365-294X.2011.05205.xV; Lohman et. al., 2016V doi: 10.1111/1755-0998.12529V; and Matz, 2018Vdoi: 10.1016/j.tig.2017.11.002V), the problem is that I do not know anyone who has personally used it so I am cautious. On the other hand, there are two main issues to take into consideration upon making this decision. Firstly, the weevils cannot be bred in laboratory conditions and are quite hard to find in the wild, therefore, the amount of RNA that will be available is still unknown and I will need to make at least 3 replicates. Also, funding is incredible low: in a year the price of an American dollar has gone from 23 Argentinian pesos to 60 which means our ability to pay (in US dollars) for the libraries and sequencing has plummeted and that situation is not likely to change in the near future. From this transpires the fact that we will to maximize the RNA extracted per ovary (on that topic I have found this protocol based on Matz (2002) and would be very grateful for your thoughts about it: http://evrogen.com/technologies/RNA-isolation.shtml), but the minimum amount of RNA per sample will also be important in choosing which method to use: for RNAseq we will need at least 500 ng of RNA per sample while for TagSeq protocols, some companies can go as low as needing 10 ng of RNA per sample. What do you think about the two methods? Is one truly better or worthier than the other for detecting differential gene expression? What would your approach be if you were in my shoes? Thank you all in advance! I am willing to post all replies... Lucia Lucia Fernandez Goya PhD Candidate Universidad de Buenos Aires Buenos Aires, Argentina luluf.goya@gmail.com Responses Hi Lucia, We did tag-seq a bunch of years ago when it emerged with the first Illumina sequencing platforms. We found a lot (and I mean a lot) of "apparent" anti-sense transcripts. Now, yes, there probably is more anti-sense transcription that we might think (I know the human cancer geneticists got interested in it; not sure if still are), but not this much. Illumina stopped supporting it, and I wonder if there were issues that they never admitted to. If you can, I would use RNA-seq. I hope this is helpful. Best, Dave Hi Lucia, It sounds an interesting project that you have. The following page is very helpful in understanding the pros and cons of TAG seq: https://dnatech.genomecenter.ucdavis.edu/faqs/when-do-you-recommend-3-tag-rna-seq/ In your case, there is no genome available for even a species in the same genus, which will be problematic. Also, you may be interested in splice variants, but the first issue is the biggest reason that TAG seq may not be the best for you. I hope the funding/economic situation gets better there. I was planning to come to Argentina for sabbatical Spring 2021, to Bariloche, but I am waiting to see given the current situation. Good luck with your project. All the best, Ben Hi Lucia, This is paper we have on BioRxive regarding our approach to do RNAseq for very cheap: https://www.biorxiv.org/content/10.1101/585810v1. There's also a pipeline to process the data, and a protocol to extract your RNA that works for very small input material (e.g. single fly head). https://lufpa.github.io/TM3Seq-Pipeline/ I developed it for my experiments where I have to sequence thousands of Drosophila melanogaster heads (tiny tiny heads) and get RNA and DNA from single heads. Take a look at it, it might help you. I'm also happy to answer any questions you might have. Cheers, Luisa Hi Lucia, I will apologize in advance for the quick response, but I think the answers to your questions are pretty straight forward. 1. Your subject heading was rad-seq vs tag-seq, but given that you're only interested in RNA you must mean traditional RNAseq (whole transcript) vs tagseq. 2. I strongly recommend that you look into RNA extraction via magnetic beads. Nice paper: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000107 My lab has moved entirely to extracting DNA and RNA via beads. Much cheaper and more efficient than columns. 3. You should probably use TagSeq. The libraries require less RNA because you're less concerned about sampling bias or amplification issues (as compared to traditional RNAseq). You also require much less total sequencing per sample with TagSeq, because you're only dedicating one library fragment per gene. You will literally save 50% just on sequencing cost along, let alone library prep. 4. You get more detailed info out of traditional RNAseq, such as splicing variation, but given the modest breadth of your project aims and budget, you wouldn't be able to immediately follow up on any of those complex issues. Good luck with the project, Jesse Hi Lucia, I saw your email about TagSeq on evoldir. I can share a bit about my experience working with TagSeq. I have recently started using the method with birds and fish. You are right, there are some tradeoffs with TagSeq vs. RNAseq. TagSeq is more affordable and you need less starting material. We have had success with samples that have concentrations as low as 5ng/ul. The library preps are less standard, so it might take some figuring out to do them yourself though there are now a few places that are offering them (University of TX Austin for example). With TagSeq, you only get ~50 base pairs of the 3' end of the gene, so it is not a good method if you are interested in sequence differences or splice variation, it really just gives you information on counts for each gene. Given this, you do need a fairly well annotated transcriptome or genome to work off of (so you can match up those 3' tags to the proper genes with some confidence). I will tell you that it does work and it is cheaper. Happy to answer more questions if I can. Good Luck, Amanda Hi Lucia, Well your title is not what you asked ;) RNA-Seq vs Tag-Seq :) We have good results with Tag-Seq, commercialized by Lexogen (Quant-Seq). The original paper protocol should be cheaper However, you need a reference genome or transcriptome, or at least a related genome/transcriptome to map the 35 bp. reads on. In comparison with RNASEQ, annotating the RNA reads is difficult if you have no reference. We used it for non model species, also with very low quantities or RNA (brain parts). See the paper : https://www.frontiersin.org/articles/10.3389/fnins.2018.00136/full Cheers, Gregory rodriguero@ege.fcen.uba.ar