Small RNA sequencing

[suludana]

Hi,
I will start with Small RNA sequencing.
Anybody can tell me how many cycles of sequencing are typically used?
Does any special indication for the analysis of data? Is there something very different from the analysis of genomic DNA sequences?
Thanks

[joa_ds]

I will be doing Small RNA sequencing in the future as well on a Solexa, but atm still installing a 454, so no real hands on experience.

But from what i have read i dont see any big difference between sequencing DNA and sRNA. When sequencing short RNAs, you basically just convert the short RNAs to DNA and then sequence the DNA (Someone correct me if i am wrong).

I know Solexa has a Short RNA kit, but the ABI SHREK kit might also be usefull.

About the cycles of sequencing... Atm the Solexa sequences sequences of approx 30-50 and 75 as a real max. I don't know what you are sequencing, but i think it will be miRNAs? they have a length of 20-25. I don't know how long the "adaptor tags" are atm, but i guess your total length should be in the range of the 30-50. In theory, with 50 cycles you should be able to sequence 100% confident...

Of course it depends on what the purpose of your sequencing is. If you want info about known sequences and just count tags or differential expression of known sequences, i would adapt my cycles to the length of your desired sequences. But if you are hunting for new sequences, i would go for 50-70 cycles. You might find a new interesting sequence...

I don't think there is something very different data-analyse wise. Just remember you need to change Ts back to Us to have the RNA sequence...

An example here: http://genome.cshlp.org/content/18/6/957

[suludana]

Hi Joa,
thank you for your response.
Yes, I will work with microRNAs. But I´m planning to use one of the recomended number of cycles of the Illumina kits (18, 26 or 36). Is it not a risk to try more?

[joa_ds]

it all depends on the lenght of the sequences you want to sequence.

Well it is never a risk to try more, it is just a waste. If there is nothing to sequence at the end of your sequence, you are basically just wasting reagent.

I'd say 36 is the safest thing to do if you want your complete sequences covered with adaptors.

This link also suggests 36 is the safest thing to do and might even be short if you are multiplexing with ID adaptor. http://seqanswers.com/forums/archive...php?t-324.html

I don't know how long the adaptors are? but a sequence with length 20-25 and adaptors added, should be around 36.

Another possibility is to run a gel in advance and take a certain cutoff, lets say 20, add adaptors and do a 26 cycle run...

You just need to avoid missing the end of a sequence because you did not have enough cycles. The sequence quality is still superb at 30, so there is no objective reason to stop sequencing (if there is still a sequence of course) at for example 26 cycles