Whenever the sequencing depth of your samples differ too much (i.e. >10X), it is recommended to perform rarefaction before normalizing your data. Note, users should also consider to remove the shallow sequenced samples as such gross difference could be due to experimental failure (i.e. performing outlier removal rather than “punish” all other samples). For more details, please refer to the paper by Weiss, S et al.
In MicrobiomeAnalyst, users can directly visualize the sample size (available on the Data Inspection page) to check the need of rarefaction for their data.