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Are unequal pool sizes for RNA-seq acceptable?
Background:
I want to find differentially expressed genes between wild type (WT) and knockout (KO) mice of mixed background using RNA-seq (Illumina HiSeq, 50M reads standard at my core). There will be anywhere between 1-4 KO and 1-4 WT animals per litter.
Because it is difficult to isolate enough cells from one animal, I need to pool cells from WT littermates and KO littermates. I need 2 animals minimum, but 3 and up is preferable. My thinking is that each litter will be one biological replicate, and I plan to sequence RNA from 3-5 litters (3-5 biological replicates). Because the number of KO vs. WT animals within a litter varies, I may have more of one genotype than the other.
Question:
If I had this scenario:
Bio rep 1: Pool of 3 WT and Pool of 2 KO
Bio rep 2: Pool of 2 WT and Pool of 2 KO
Bio rep 3: Pool of 2 WT and Pool of 4 KO
...or any combination thereof, would this be a statistically poor design? Should I keep the number of animals in each pool constant for all biological replicates? Only within biological replicates?
Another consideration: Will this be representative of only one mating cage? Cost/time is limited to maybe one time on the sequencer, two chips.
I want to find differentially expressed genes between wild type (WT) and knockout (KO) mice of mixed background using RNA-seq (Illumina HiSeq, 50M reads standard at my core). There will be anywhere between 1-4 KO and 1-4 WT animals per litter.
Because it is difficult to isolate enough cells from one animal, I need to pool cells from WT littermates and KO littermates. I need 2 animals minimum, but 3 and up is preferable. My thinking is that each litter will be one biological replicate, and I plan to sequence RNA from 3-5 litters (3-5 biological replicates). Because the number of KO vs. WT animals within a litter varies, I may have more of one genotype than the other.
Question:
If I had this scenario:
Bio rep 1: Pool of 3 WT and Pool of 2 KO
Bio rep 2: Pool of 2 WT and Pool of 2 KO
Bio rep 3: Pool of 2 WT and Pool of 4 KO
...or any combination thereof, would this be a statistically poor design? Should I keep the number of animals in each pool constant for all biological replicates? Only within biological replicates?
Another consideration: Will this be representative of only one mating cage? Cost/time is limited to maybe one time on the sequencer, two chips.
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