Adapter artifacts 16S library prep from low density samples

[desertwillow]

Hi there!

I'm new to the world of library prep for MiSeq thought I would reach out to the community for some suggestions. We are preparing 16S libraries from low density microbial communities with primers designed for the V1/V3 hypervariable region of 16s rRNA followed by barcoding. We are having problems eliminating the adapter artifact peak ~130 bp (usually 4x greater concentration than our 300 bp product) even after bead size selection. Anybody know any tricks for eliminating the artifacts?

[nucacidhunter]

You have not given enough information considering many protocols which are used for 16S diversity profiling. I assume you are using primers for amplification not adapters for ligation. From your description that 130 bp fragment could be primer-dimers. They are prevalent when the target region is not amplifying efficiently at least under three conditions: 1- low concentration of target sequences in reaction, 2- non-optimal annealing temperature and 3- primer design where primer heterodimer interactions are stronger than primer-template. If the aim is to reduce them in first place, attention should be paid to eliminate or reduce above mentioned causes. Sometimes reducing primer amount also helps. If you are getting enough 16S amplicon with expected representation of community, they can be washed away by cleaning amplicons with 0.9-1x AMPure beads. You might have to do bead wash twice if they are persisting through first wash. That should not affect your target amplicon concentration much. Last resort is gel cut either manually or by instruments. Following link also has a nice presentation:http://www.corning.com/uploadedFiles...20Solution.pdf