I used mothur for the analysis of my 16S rRNA and ITS2 dataset from soil samples. Mothur team especially Sarah was kind enough to guide me throughout my analysis. I have submitted my manuscript to the journal and received comments from the reviewer. One of the comment is related to the number of OTUs and his statement is given below,
“There also seems to be problems with the quality of the bacterial reads- the fact that the rarified to 3,500 when they had 75K + reads. So only being able to identify a few hundred OTUs of bacteria is very low- my experience is that you should get 5000+ OTUs”.
When I normalized the data then my smallest sample has about 3500 sequences ultimately, number of OTUs were also very low. Even without normalization there was a not a big difference in number of OTUs. While reviewer has issues that with 75K + reads why are the number of OTUs in hundreds? Moreover, Chao1 estimates OTUs almost double than the observed OTUs. In contrast when I used ITS2 dataset, the number of OTUs were more compared with 16S. Please help me to reply the reviewer about his question regarding less number of OTUs instead of his rough estimate of about 5000+ OTUs from same soil samples.
Your quick response in this regard will be highly obliged.