Amount of starting material for DNA library prep

[jport]

Hi there,

I had a question regarding the amount of starting material going into a DNA library prep for MiSeq. I am using a KAPA library prep kit for Illumina and usually aim for around 150 ng of DNA per sample going into the prep.

Up to this point I have been building individual libraries for every sample, and then pooling the libraries in equimolar concentration for sequencing. We just switched our procedure though and are now ordering our PCR primers with a 6 bp tag identifier at the 5' end for each sample, so that after PCR each sample has a unique tag ID that can be used downstream to separate the samples after sequencing. We then pool all the tagged amplicons in equimolar concentration and build a single library for sequencing.

My question is, when pooling the tagged amplicons, should I add 150 ng of each sample to the pool and then use 150 ng of the pool to build the library or can I add less than 150 ng per sample (say 20 ng per sample) and then use 150 ng of the pool for the library? My concern with the latter is that when pooling many samples (~50) the mass of DNA per any individual sample in 150 ng pulled from the pool will be very low (~3 ng) and I'm not sure if that's ok considering what's lost during the library prep process and how much is needed for sequencing.

Any insight is appreciated!

Thanks,
Jesse

[nucacidhunter]

Could you provide more info on your library prep method. It seems to me that you are amplifying some target regions by PCR with 5' tagged primers then you prepare sequencing library by A tailing, adapter ligation and PCR amplification.

[lihzhou]

I think i get your idea! that is just decided by your quantification method of the PCR ampllicons, not the amount. Two suggestions: 1) the concentration you write down should be within the precise measurment range; 2) the volume of each amplicons pooled should large enough to escape equipment error of pipettes.

These will help you get equal reads number for each amplicon in final seq-data.