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11-Aug-2018 New paper describes octave plots for visualizing alpha diversity.

12-Jun-2018 New paper shows that one in five taxonomy annotations in SILVA and Greengenes are wrong.

18-Apr-2018 New paper shows that taxonomy prediction accuracy is <50% for V4 sequences.

05-Oct-2017 PeerJ paper shows low accuracy of closed- and open-ref. QIIME OTUs.

22-Sep-2017 New paper shows 97% threshold is wrong, OTUs should be 99% full-length 16S, 100% for V4.

UPARSE tutorial video posted on YouTube. Make OTUs from MiSeq reads.



Long overlaps are not needed, so 2x250 can do better than V4

Some people have suggested that the almost complete overlap obtained with 2x250 V4 is a good strategy because it gives the best error correction. I disagree. In my opinion, for most applications the reduction in error rate is not dramatic and does not compensate for the improved phylogenetic resolution you get with longer sequences.

Using UPARSE or UNOISE gives very accurate OTUs regardless of the overlap length. As a proof of concept, you can generally get good OTUs if you throw away forward reads for V4 and use reverse (R2) reads only, despite the lower quality of the R2s. I therefore recommend sequencing the longest possible amplicon that will give a minimum overlap of around 20 to 30 nt.