Failed Sample QC

[Kates106]

Hi all,

I am a rookie in terms of NGS and am currently pursuing a project in Puerto Montt, Chile on microbial diversity and community composition. Due to lack of resources, Macrogen (in Korea) is performing the majority of the bioinformatics-preparation of amplicon libraries and sequencing. With a total of 18 samples, I was recently and unfortunately informed that 5 samples failed, with "no peak" in the Agilent bioanalyzer & Caliper Labchip GX DNA High Sensitivity portion of the project. I have attached the failed and successful results (three attachments) and am curious if anyone has any advice as to 1. what went wrong and 2. what could be the next step with these failed samples. Any help/advice would be appreciated, thanks.

Katie

[trexbob]

Hi Katie,
Were the five that failed somehow different from the others as far as what they were from? Is there a possibility that those five somehow had a chemical present in the isolated DNA that could prevent amplification? Diluting the template DNA further sometimes helps with those situations. I would try either new DNA preps or further dilutions of the template DNA going into the PCR reaction (making the amplicon) until you can reliably get a PCR product that looks ok on a gel and go from there...

[Kates106]

Hi Trexbob,

Thanks so much for your reply. The DNA was extracted from sediments, so there is definitely a possibility that the samples may contain inhibitors. I purified all 18 samples, but perhaps these five need a second purification? Two of the five failed samples are replicates from the same site; whereas, the other three are from different sites.

I will try diluting the template DNA first and go from there...

Thanks again