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RNA seq Newbie needing help
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2.9 years ago
Ratgirl89 • 0
Ireland

hello,

I am a PhD student planning on doing an RNAseq experiment looking for gene expression changes in Salmon fish. I am planning on 8 tanks of salmon in total. 4 tanks will be infected with a disease and 4 tanks will be healthy. I am planning on taking samples from the fish at 9 different time points. I plan to take Gill, Kidney, Liver and Blood samples.

Per time point I plan on taking 2 fish from each tank. Therefore 8 healthy and 8 unhealthy fish. 16 in total. Taking 4 samples from each fish would equal 64 tissue samples per time point. So at the end of all my timepoints I would end up with 576 sample, 288 healthy samples and 288 unhealthy samples . This is where Im getting really confused. Do i have enough replicates.

How do I know my sample size is big enough, can someone recommend a power analysis software.

What should I ask for when looking for quotes from companies. As budget is an issue I need to keep that in mind. I know I can pool libraries but not samples.

What depth and number of reads is recommended for looking at gene expression.

How many samples can be put on each Lane.

If anyone can help me i would be really thankful.

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Will you be sampling the same fish (somehow marked) over the time period? Are the fish going to be artificially infected? What is the significance of 9 time points? How long will this experiment last?

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I am looking at the early immune response so 9 points were just an example really . Days 0, 1, 2, 3, 5, 7, 10, 14, 21,. The fish sampled will be euthanized at each time point. There will be approx 30/40 fish in each tank at day 0. They water they are in will be infected with a parasite (This will brought in fresh from a sample and will then be grown up in lab) , and i will be examining the progression of the infection. Experiment will be stopped at 21 days.

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Do you expect a lot of variation from one fish to another within the same tank? (Any first test by qPCR on a set of genes that would likely change due to your infection?)

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18 months ago
bernatgel ♦ 1.9k
Barcelona, Spain

Performing an experiment with 576 samples without at least a small pilot seems quite risky. At least to check that there are some changes and that you can find the what you would clearly expect.

I think something like a pilot with two tanks, 2 time points (0 and 7), 2/3 fish per tank and a single tissue (up to 12 samples) could help you evaluate the total change detected and the differences between individuals of the same group.

We've been bitten before by an RNA-seq experiment with large differences in the control group (heavy batch effects) and we ended up with basically unusable data.

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