Hi everyone,
I have a question about pooling libraries for RNAseq.
I have 12 samples that I would like to run RNAseq with. Samples 1-6 were extracted from pure fungal cultures, however, samples 7-12 were extracted from fungal cultures that were contaminated with plant tissue, bacteria, and protozoa. I am specifically interested in the gene expression of the fungal tissue. I was wondering, because of the contamination in samples 7-12, is it possible to unequally pool the prepared libraries during RNAseq? ( increasing the amount of RNA libraries of samples 7-12 to increase the sequencing depth of the fungal RNA in the samples). Would that be acceptable? Would it give me any trouble when analyzing the data? ( My goal is to compare gene expression between samples 1-6 and samples 7-12. )
Thank you for your time and help!
Hi Jing,
since you already prepared the librairies, I won't do that, especially if you want to run DEseq analysis. Since all samples will be sequenced in same run, the amount of relevant RNA will be equal in both groups. the only thing is that you'll have more untarget sequences in one of the group, the trash will be higher in that group.
what you should do would be to take into account the amount of contaminant before doing the librairies and if you know the proportion of it take proportionaly more total RNA for group2, to be sure you have equal target RNA for all samples in both groups.
fred
Frederic Lepretre
Hi Fred,
Thanks for your kind reply!
I haven't prepared the libraries for individual samples. It's hard to know how much contamination there is. But, if I proportionally pool more RNA for group 2 during sequencing, would it give me any bias in the experimental design?
Actually, another goal of this project is to compare the gene expression between samples 1,2,3,7,8,9 and 4,5,6,10,11,12. Would I be able to correct the data based on the numbers of total reads for each samples during data analysis?
Thanks,
Jing
Mathew A Beale
Hi Mathew,
Thank you for your reply!
I have not prepared the libraries for each individual sample yet. The non-fungal RNA comes from various microorganisms and plants, I guess it's not that easy to accurately estimate the percentage of contamination. My feeling is ~60-70% RNA from samples 7-12 is targeted RNA.
I was wondering if increasing the amount of RNA libraries of samples 7-12 in a pooled RNA library would be a good idea to increase the sequencing depth for samples 7-12?
And yes, I will map reads back to a reference genome/transcriptome after I get the sequence data. I would like to compare DEseq between group 1 (including samples 1-6) and group 2 (including samples 7-12), and in the meanwhile, I would like to compare samples 1,2,3,7,8,9 (as a group) and samples 4,5,6,10,11,12 (as a group). Would unequally pooling the libraries introduce any bias in my future data analysis? I thought if I am able to map the reads back to a reference genome and correct the data based on the numbers of total targeted reads for each sample, I would be fine. I may have not seen the whole picture. I would really appreciate it if you could share your thoughts on it.
Thank you for your time and help!
Jing
Mathew A Beale
Hi Mathew,
Thanks for the comments!
I am thinking about using NextSeq 500 v2 75 bp PE which will generate 400M reads, on average each sample will get 33M reads if I pool 12 samples equally. And the fungal genome is ~80Mb.
I don't have any experience with RNAseq or DEseq. Could you recommend a package that can be used to normalize the data by the numbers of total targeted reads?
Thank you,
Jing
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