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Old 03-13-2014, 09:10 PM   #1
capsicum
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Join Date: Jul 2012
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Default Illumina 16S libs - tech & QC questions about construction and data comparison

I wanted to ask for people's opinions and experiences:

I'm looking at making some Illumina-compatible sequencing libraries. Illumina has a two-step PCR method where the first PCR amplifies a region of the 16S gene and the second PCR adds adapters.

1. Does anyone carry out the first and second PCR steps together on the one reaction? I read about someone doing this, but I can't seem to find it again.

2. What QC do you do on your primary and secondary PCRs? How do you deem a success or failure? Just band visibility on a gel?

3. How do you deal with a project where some samples show very strong amplification and others showed only just sufficient amplification for sequencing? Do you optimise the PCR (by diluting the template, for example) to increase amplification of the poorly performing samples, or just sequence them anyway if there's enough product to do so?

4. If you dilute your samples to obtain better amplification, do you think this needs to be done for all samples that will be compared? I.e. do you think it will bias the results if you sequence samples that have been treated differently in this way and then compare them? If not, then do you think it's legitimate to compare samples that have radically different amplification efficiencies?

5. Do you think it's important to normalise the input DNA concentrations for samples that will be compared? What about the input source material? We have some projects where samples that need to be compared might contain DNA concentrations that differ by 100x or more.
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