Question: Normalizing small RNA Seq Reads
0
gravatar for empyrean999
5.2 years ago by
empyrean999170
Canada
empyrean999170 wrote:

Hello All,

This could be a easy solution but just wanted to get your opinion on this. We have 10 different small RNA samples sequenced for arabidopsis. They are varied in read numbers from 10 million to 30 million . We wanted to look at reads mapping to 5' end of a gene. I used that gene as reference and mapped all the reads and got read counts mapping to it.

So to get relative expression, can i use simple normalization using total reads as my reference is only one gene or should i use proper techniques such as TPM or TMM ?

ADD COMMENTlink modified 5.2 years ago • written 5.2 years ago by empyrean999170
what is exactly your goal? assessing differences in the 5'UTR coverage of that gene?
ADD REPLYlink modified 5.2 years ago • written 5.2 years ago by Martombo2.7k

The goal is to understand difference in num of reads mapping to that 5' region of gene approx (150bp) . REads only map to that part and no reads seen in rest of the gene. i have read counts only for that 150bp location . I wanted to see across different samples, what are the normalized read numbers ?

For examples

Sample name Total No of reads Reads mapped Normalized num
1 100 5 5/100 = 0.05
2 200 20 0.1
3 1000 50 0.05

Would the above nomalization enough in my case or is there anything i am missing?

ADD REPLYlink modified 5.2 years ago • written 5.2 years ago by empyrean999170

you said you have small RNA samples right? if so, you would actually de-rich mRNA sequences in the process (even with size selection only) and for this reason, looking at a small window of a gene would not give you a reliable indication of a biological process. or are you actually making the hypothesis that the reads you see on the gene would come from a small RNA that would target the gene?

ADD REPLYlink written 5.2 years ago by Martombo2.7k
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