18S cluster.split VS phylotype


I'm trying to work with some amplicons of 18S, I follow the MiseqSOP, the thing is that I get to much chimeras, and lost a lot of sequences, almost the 60%, probably this means I did something wrong in the PCR, any way I get untill cluster split, get the OTUs and everything, and the first weird thing I see, is that I have a good coverage between 88-99% but the rarefaction curves look horrible. I try to do the phylotyope approach to be able to use more sequences, I got good curves but the number of OTUS was like 10 times less than when I use cluster split. When I analyze the b-diersity and everything i got almost the same results with the diferent approachs, the thing is I don't now if use one approach is better, use cluster.split (using like one million of sequences) or Phylotype(using 4 millions of sequences). Both gave me the same results regarding to the community.


Sorry you’re having problems with analyzing your data. Could you layout your problems one at a time and provide the commands you are running? I suspect some of your problems are related to earlier problems.

Are you losing 60% of your unique sequences or total sequences? I think 60% of your unique sequences would be reasonable. If it’s the total number of sequences then I would wonder about your PCR. How many rounds are you doing? How long is your extension time? We encourage a 5 min extension time since that has been shown to limit the number of chimeras.