I am a newbie for NGS.
I am doing rna-Seq. My aim is to compare gene expression between samples.
Question 1: I did single end 50 bp sequencing for a bacterial strain. Each sample got ~10,000,000 reads. For most samples, ~10% reads are rRNA which I think it OK. But for several samples, ~66% reads are rRNA. It means only maximally ~3,400,000 reads are useful. Do you think if ~3,400,000 reads are acceptable or the number is too low?
Question 2: Since different samples have different rRNA proportions, I cannot directly compare gene expression using the rest reads, right? How should I do normalization?
Questions 3: I have tried my best to using same amount of cells, RNA, and cDNA during sample preparation and I got approximately same amount of reads between samples. But, it is still possible to get bias between samples. Should I do normalization against a housekeeping gene by assuming the housekeeping gene has the same expression between the samples?
I am doing rna-Seq. My aim is to compare gene expression between samples.
Question 1: I did single end 50 bp sequencing for a bacterial strain. Each sample got ~10,000,000 reads. For most samples, ~10% reads are rRNA which I think it OK. But for several samples, ~66% reads are rRNA. It means only maximally ~3,400,000 reads are useful. Do you think if ~3,400,000 reads are acceptable or the number is too low?
Question 2: Since different samples have different rRNA proportions, I cannot directly compare gene expression using the rest reads, right? How should I do normalization?
Questions 3: I have tried my best to using same amount of cells, RNA, and cDNA during sample preparation and I got approximately same amount of reads between samples. But, it is still possible to get bias between samples. Should I do normalization against a housekeeping gene by assuming the housekeeping gene has the same expression between the samples?
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