Suggestion regarding reads overlap and paired end sequencing

Hello, Qiimers,

I would like some advice on my sequencing protocol. I normally use V1-V2 for my 16S sequencing; however, I plan to go V3-V4, as per the standard Illumina protocol for my next project. For what I understand, I should expect a ~460nt PCR product. I was wondering if sequencing 250x2 would be sufficient (40 nt reads overlap), or if you would rather suggest going 300x2 (140nt overlap). The former seems sufficient in my opinion, but it is the first time I use this region, and I would like to hear your opinion.


If it is an option, I would go for 300x2, since in my personal experience and based on questions on this forum there are a lot of cases when retained after primers removal and quality trimming overlapping region was not sufficient to merge bacterial reads with longer V3-V4 region, so basically datasets were biased towards bacteria with shorter targeted regions. Otherwise, I would proceed with V1-V2 or V4 regions.

PS. It is only my opinion.

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