Aligning Cyanobacterial ASVs with Cyanoseq Reference Using MAFFT in QIIME 2

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Hello Everyone,

I am working with 16S rRNA V4–V5 amplicon data and have filtered my feature table and representative sequences to retain only cyanobacterial ASVs (phylum Cyanobacteriota). I would like to align these sequences to the Cyanoseq reference database using MAFFT, and later construct a phylogenetic tree with IQ-TREE.

However, I am unsure how to go about the alignment using a reference within QIIME 2. I reviewed the MAFFT plugin documentation, but it seems that it only supports aligning a set of input sequences to each other, not to an external reference alignment or set of reference sequences.

Could you please advise on:

  • Whether aligning to a reference database like Cyanoseq is possible using the MAFFT plugin in QIIME 2?
  • If not, whether it would be better to merge my ASVs and the reference sequences and perform a joint alignment instead?
  • Any recommended approach to integrate external reference sequences into the alignment and tree-building process?

Thank you for your guidance.

Best regards,
Anna

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Hi @anna.abbey,

If you run qiime alignment mafft-add --help you should be able to do what you need. Then you can run qiime phylogeny iqtree ... or qiime phylogeny iqtree-ultrafast-bootstrap ...

-Mike

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Hi Mike,
Thanks. I will try this and let you know if it works.

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I forgot to mention, if you do not have an alignment you can also make use of qiime feature-table merge-seqs ... to combine unaligned reference sequences and your ASVs. Then you can align them, and construct your phylogeny. There is also qiime feature-table merge-taxa ... if needed.

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okay thanks! well noted

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