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Old 03-03-2016, 10:11 AM   #1
urchin
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Location: California

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Default Help with primer design for 16s

I am another newbie trying to get through a first project in NGS.

We are trying to amplify universal prokaryotic sequences for 16s rRNA gene (V3-V4 region) from benthic marine critters - mostly from microbiome extractions using materials from invertebrates and some algae. I was told that using the Fadrosh et al. 2014 dual-indexing method would improve our results a lot when we do PE250. Hopefully that is true - yes? (No?)

Also, right now all the current primer sets are going through continued evaluation and most seem to be found lacking in one way or another. Does anyone have a strong opinion on choices based on actual experience with getting good results? Has anyone tried the primers discussed in Takahashi et al. 2014, Pro341F/Pro805R? Did they work as well as claimed?

Thanks for any help pointing me in the right direction!
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Old 04-29-2016, 01:04 PM   #2
urchin
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Hello All,
Since nobody answered me I'll at least give some advice to anyone else who reads this post by searching the topic on the site.

I found that Silva TestPrime can really help to narrow down some of the primer choices, especially if you want to find a more universal primer set for Prokaryotes versus one that hits only Bacteria or Archaea. Also, the information includes what type of research projects the primer set has been used in before and scored that organisms (e.g. marine metagenomics).
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