Low input/adaptor dimers

[whw]

Hi all,

I'm preparing libraries from very small amounts of RNA, around 100-1000x less than the 1ug recommended by illumina. Generating a "library" seems easy but on analysis it turns out to be mostly adaptor. Has anyone experimented with adding less adaptor? Or any other ways of optimizing library preparation for small amounts of nucleic acids?

I get the impression this is a fairly common problem, I'll be on the phone to illumina in a bit and I'll pass along any gems they give me. In the meantime what do all you seqanswerers think?

WW

[arolfe]

I believe that different Illumina kits used different adapter concentrations. For example, I think the only difference between the ChIP-Seq and genomic (?) kits was a 100X difference in adapter concentration. So yes, it makes sense and you should be able to look around to get an idea of where to start.

[HESmith]

The ratio of adaptor to sample is probably the most critical variable in library construction. You should be able to adapt the ChIP-Seq protocol, which works with 10ng dsDNA. You may need to perform an additional round of size selection to remove adaptor dimers. Alternatively, you can perform linear amplification of your starting material.

Harold

[whw]

I have the Ultra-low input mRNA Seq Guide from Illumina which is optimized for 2ng of dsDNA. I'm thinking I'll try their adaptor dilutions and see what happens, hopefully the ligation chemistry won't be too different.

[griffes]

what adapter dilutions are you using for RNA?

[ETHANol]

Just in case you were wondering from information I've gathered from other threads the concentrations of the TruSeq adapters are:
DNA Sample prep Kit adapters = 15 μM
RNA Sample Prep Kit adapters = 0.25 μM

And yes, adjusting the amount of adapter to the amount of DNA you want to ligate it to is good practice. The adapter should be in excess but I'd say not more then 10 fold and probably better to be less.

[whw]

what adapter dilutions are you using for RNA

The ultra-low input protocol suggests a dilution of 1:19 for 1-4ng of sample. I thought I'd start in that range.

Just in case you were wondering from information I've gathered from other threads the concentrations of the TruSeq adapters are:
DNA Sample prep Kit adapters = 15 μM
RNA Sample Prep Kit adapters = 0.25 μM

And yes, adjusting the amount of adapter to the amount of DNA you want to ligate it to is good practice. The adapter should be in excess but I'd say not more then 10 fold and probably better to be less.

That's very helpful, thanks for the tip Ethan(ol)