Hi QIIME2 community, My colleague @Lauren I have been using the Ion torrent 16S Metagenomics Kit to sequence some clinical samples that we have. Our sample sets are both longitudinal. The kit consists of two primer sets, one for V2-4-8 and one for V3-6,7-9. The products of the reactions are pooled …

This might be common knowledge already, but I wanted to articulate why I think close-ref OTU picking could be a good fit for multi-region-studies Three high-level strategies for defining OTUs... are canonically described as de novo, closed-reference, and open-reference OTU picking... Each of the…

[image] colinbrislawn: Modern ASV methods aim to be just as consistent without introducing database bias, but for this project we are knowingly using this strong bias to normalize across regions. Thanks for clarifying @colinbrislawn ! I agree, and I also generally avoid closed-ref OTU clusteri…

Hello to all, I'm trying this pipeline on my data. This is the step (as suggeste above): 1- Import data 2- demux 3- dada2 denoise-pyro 4- qiime vsearch cluster-features-closed-reference 5- qiime fragment-insertion sepp In the last step (5) I had an error message... This is the script that I…

Hi Everyone! @cjone228 and I have another couple of points we would like clarified: Our fastq files contain single-end mixed orientation reads (both forward and reverse). We imported our data using SingleEndFastqManifestPhred33V2 according to the QIIME2 the Importing Data Document. However, we…

Oops! I meant to thank @Nicholas_Bokulich , @colinbrislawn , and @jwdebelius for your clarifications on OTU clustering, SMURF, and phylogenetic analysis above - all of your posts were extremely helpful! Lauren :qiime2:

[image] Lauren: Our fastq files contain single-end mixed orientation reads (both forward and reverse). We imported our data using SingleEndFastqManifestPhred33V2 according to the QIIME2 the Importing Data Document. However, we recently noticed that the Importing Data Document states “In this va…

Thanks Nicholas for your correction. Now I'm a little confused about how to continue... I have to download the tree from Greengenes' database and then how do I continue? Can I perform in the next step the core metrics? qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza …

Hi @rparadiso , [image] rparadiso: –i-phylogeny rooted-tree.qza (green genes tree?) Yes just import the tree that corresponds to the reference sequences that you used (e.g., if you used the 99% ref, use the 99% OTUs tree) as a Phylogeny[Rooted] following the import instructions. If you run i…

Thanks for the feedback @Nicholas_Bokulich ! [image] Nicholas_Bokulich: Your reads are all forward or reverse because in this case F/R mean the read direction on the sequencing instrument, not the orientation respective to the genome (which is mixed in this case). So you are doing the right thi…

We may have just answered our question about closed reference OTU clustering - in the QIIME2 documents for closed reference clustering of features, the parameters section has the following option: --p-strand TEXT Choices('plus', 'both') Search plus (i.e., forward) or both (i.e., forward and rever…

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Possible Analysis Pipeline for Ion Torrent 16S Metagenomics Kit Data in QIIME2?

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