Diversity core metrics of Sanger sequencing data:

[Nicholas_Bokulich]

Hi @Masanobu_Hiraoka,
Thanks for sharing the data and commands — this allowed me to track down the error.

It looks like the issue is not due to the tree-building pipeline, but rather a known bug related to the use of dereplicate-sequences without further clustering.

This issue — and possible workarounds — is also described here:

Mismatched feature IDs

Thanks for sending the viz in a DM! Okay, guess what? Even though the viz looks okay, if I pull out the FASTA file from within: >3cafeb270ab9f7183bbc2e7c24b7cc1ffb2f196c UU3micro-18S-12_S14_L001_404900 CCTGAAAGCCGGTAATGACTTTCTCGCGTCAAACCGCGAAAAGCCAGGCGTGACCGAACTCCTCAGCGGACTCCAGTACGAAGTGATCCACATGGGTGACGGAGCCAAACCCTGGCCCACCAGCAAAGTGACCTGCCATTACCAT >bbfdbd5a45738494ef2a3fc5f95a878bfb9f8475 UU3micro-18S-12_S14_L001_404902 ATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGACTGGTTCATCGAGCTTACAACCT…

Using cluster-features-de-novo should get you moving, even if you cluster at 100% similarity.

@Masanobu_Hiraoka

And I might have another problem, “why there are so few reads per sample in my dataset”. The highest # of reads in one sample is 139.
Is this supposed not enough to run any typical microbial diversity analyses?

This is an unrelated issue. I do not see anything in the QIIME 2 part of your pipeline that would cause this issue (since you are just dereplicating the input sequences, no filtering involved), so I think you are right to look to the qiime1 forum to solve this issue... if you rule out qiime1 as the issue, please open a new topic to get help for this issue. Thanks!

Good luck! I hope my workaround proves suitable to you.