We Need Advice On Choosing The Appropriate Parameters For Smallrna Sequencing

Question

[sethugunja]

Hi, We would like to do the small RNA sequencing to look at the non coding RNAs profile in patient versus normal conditions. Our goal is to

1. identify the differentially expressed ncRNAs in patient versus normal and
2. identify the total adundance of the small RNAs and poly(A) abundance of the small RNAs(as there were some ncRNAs having poly(A) tail )

As we are very new to this field, we would like to know the things we should take care of before going for sequencing like

1. Read length
2. paired or single end
3. coverage
4. depth of the sequencing (to identify low abundant small RNAs)
5. How many reads are required

Are there any other things that we should think of ?

Any suggestions or comments would be appreciated.

Thanks Sethu

Answer 1

[Nick]

Your questions are a bit too general. What is the NGS technology used at your site? Illumina would be my guess as it's dominant right now but it's not the only game in town.

Assuming Illumina, here are my two cents (I am not an expert on short RNA):

1. Read length - as short as possible. You are aiming for short RNA after all. Nowadays with Illumina the shortest read length that you would encounter in practice is 40. This shall do.
2. Single end
3. What do you mean by coverage?
4. That's hard to say. Deeper sequencing would add statistical power - but up to a point. You get more by adding more replicates. The problem is that the replicates nowadays are more expensive (not to mention more cumbersome or, sometimes, even impossible) to produce than to throw more sequencing depth in. So, hard to tell really.
5. Again - too general. The more the better - but this follows anyway from the chosen sequencing depth.

[sethugunja]

Thankyou for the info. Yes, we have illumina facility at our site. 3. Coverage I meant here is the sequencing coverage with which the seq has to be run.