ddRAD library low concentration

[dBlatt]

Greetings,

I am making a ddRAD library and post-digest and cleanup my concentrations are very low. Since I have to standardize my DNA concentrations pre-PCR I would have to standardize at a very low mass of DNA (10ng per sample) to get the majority of my sequences standardized. If I am for a higher mass of DNA per sample (20ng) I won't be able to meet that level with the majority of my samples.

My question is: is it is better to have more standardized samples at a low DNA mass or less standardized at a higher DNA mass?

Thank you!

[TKC]

I am somewhat new to ddRAD BUT- If it were me, I would aim for higher standardization. With less standardization, you further bias the composition of the final library, meaning higher loci dropped out due to low coverage across samples (although, depending on your application this may be more or less important to consider??)... And while there are other factors causing variation in the per-sample contribution of the final library I would think it best to minimize contributors wherever possible...

In our lab, we typically use 100 ng total input per sample going into ligation (followed by pooling of samples, size selection, etc...), and generally only use 1/4 - 1/2 of the size-selected and pooled library for the PCR, and of this we generally have still more than needed for sequencing.

I am trying a library prep now where I am in a similar position with very little DNA available per sample (~20 ng AFTER digestion and ampure cleanup), and will let you know how it goes (I'll know sometime next week- maybe someone else with experience will chime in), but I suspect that I will still have more than enough in the end. You could also add additional cycles to your PCR, but this comes with the cost of higher error, etc...

Also, out of curiosity how much DNA did you have going into the digestion?

[TKC]

Hi again,

I just realized that the original post was fairly old (and, likely you have figured out your library prep issues?), but for completion's sake I'll add that even in my recent library prep with very low input DNA (~20 ng per sample after digestion + cleanup), we still have WAY more DNA than is necessary for submitting to the core facility, without adding PCR cycles.

[Gulzar Khan]

I have the same problem of lower DNA quantity?

Hi again,

I just realized that the original post was fairly old (and, likely you have figured out your library prep issues?), but for completion's sake I'll add that even in my recent library prep with very low input DNA (~20 ng per sample after digestion + cleanup), we still have WAY more DNA than is necessary for submitting to the core facility, without adding PCR cycles.

Can you please share the protocol. Thanks in advance.