Hello everyone, My colleagues and I are doing the analysis of some low biomass samples with Qiime 2 (v2021.4). It's the first time that we do a microbiota analysis and we have found some strange results. In our Feature table we have a total of 56 samples, 4 commercial mock community replicas and so…

Welcome to the forum, @Sergio_Garcia_Segura ! [image] Sergio_Garcia_Segura: 50% unassigned. This often indicates that your sequences are in mixed orientation (both forward and reverse together). Take a look at the RESCRIPt tutorial . The orient-seqs command will align your sequences to a refer…

Hi @ChrisKeefe ! We have tried to orient the sequences with the RESCRIPt tutorial that you have suggested and it seems that the problem has largely diminished. Now, the unclassified are only 1% and those classified as 'k_Bacteria' are approximately 5.5%. It's a great improvement over the previous si…

Glad to hear that helped! [image] Sergio_Garcia_Segura: Even so, the results are not exactly as expected in the mock community Not sure if there's a question here - if there is, please open a new topic for it. [image] Sergio_Garcia_Segura: have also had the doubt that if it is true that…

[image] ChrisKeefe: Not sure if there's a question here - if there is, please open a new topic for it. Don't worry, I have some ideas to try to improve it. If I get in trouble I will open a new topic. [image] ChrisKeefe: Are you asking whether your original diversity analyses should be r…

Your diversity metrics have been impacted, and you should re-orient, dereplicate, and then re-run. Two examples for you to consider: observed-features is a count of the number of unique features in your data. If a feature is present in your data in both "forward" and "reversed" orientation, it woul…

Awesome! Your explanation has helped me better understand the process and my mental scheme of how the sequences are in my samples. You have been a great help, thank you very much for everything! We will implement what you just suggested. I feel that now we are closer to having a satisfactory analys…

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