Hi All,
I want to do RNA sequencing from single and mixed species biofilm. Basically, I grew three different bacteria and combination of them (4 samples in total) then isolated total RNA from the biofilm cell. As I do not have equipment for library preparation, I am planning to submit the total RNA and let the sequencing company does the library preparation (cDNA synthesis and indexing). However, the library preparation is a bit expensive. I was just wondering whether it is okay instead of doing library prep for each single bacteria, I want to pool samples from the singles bacteria to have one single index so instead of having 4 samples (3 single and 1 combination), I will only have 2 samples (1 pool and 1 combination). This will dramatically reduce cost of library preparation as I will run 3 replicates per sample. FYI, I have whole genome sequenced all of bacteria used in this study as reference for RNASeq.
You might be able to get away with pooling the samples if they are different organisms and you have multiple reference genomes to align your reads. I am not sure how you will handle conserved sequences.
Since you are studying biofilms, tools developed for meta-transcriptome analysis may allow you to do certain things. See these papers, Article Urich TA, Lanzen J, Qi DH, Huson DH, Schleper C, Schuster SC...
Article Microbial community gene expression in ocean surface waters
How about conventional multiplexing of sequencing runs with unique indexes? This can also reduce the cost of sequencing, rather than waste money generating uninterpretable results.
Wisnu Wicaksono
Sure RNA-seq is an expensive technique that is why it is not so common. Designing a proper experiment is as important as any other part in RNA-seq. So, if you have limited budget, design your experiment in a way that you will have maximum utilization of your experiment and data.
Pooling all the biological replicates without indexing is just waste of valuable information. And what does genomic information will tell you in RNA-seq? Its just reference and will help you identify what? And RNA-seq is done to check what and HOW MUCH.
Nevertheless, if there is absolute need to reduce the samples, remove the technical replicates from RNA-seq and keep all biological replicates. That would be a best trade off.
Chinedu Anthony Anene
I am not really sure what you wanted to say in your comment? If he is pooling same bacterial triplicates, where does the sequence conservation comes into play? And what is alternative for the multiplexing with unique indexes, he is also mentioning the library prep and pooling with unique indexes?
Thank you all for your response.
Following Abhijeet Signh´s reponse, I am not planning to pool the biological replicate. I am planning to pool the three-single bacteria sample together. Therefore instead of doing library prep for three single bacterial RNA (let say bacteria A, B, and C - I grew them individually) and mixed of them (I grew A, B, and C together) - in total will be 4 samples. I will pool RNA from bacteria A, B and C together (per replicate) and do a single library prep so in total I will only have 2 samples (the pool and the mixed). I thought if it is possible, It would dramatically reduce cost for library prep due to I will have three replicates per treatment.
Again, thank you all for your insight. I do really appreciate it.
Wisnu Wicaksono
Even though they are different bacteria, they share almost 95 % of the genome, so it would be totally impossible to figure out which bacteria had what kind of expression profile. Do you get me?
Abhijeet Singh
I interpreted the original post as pooling different organisms, which is the case. I do agree with your comment on designing the experiment properly.
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