Aligning predicted 16s Sequences

Dear Professor,
May I ask a question? For the reads obtained from metagenomic sequencing and then predicted Tthe 16S DNA sequences, is it necessary to align these sequences with the V3-V4 or V4 regions of 16S RNA, and only those meeting certain threshold can be used as input files for qiime2? If there is no prior alignment with the V3-V4 or V4 regions of 16S RNA, and instead the predicted 16S DNA sequences from metagenomic reads are directly input into qiime2 for clustering into OTUs, taxonomic classification, and diversity analysis, would this be reasonable?
I am eagerly awaiting your guidance, thank you!

Hi @li0604,
Could you give us more information about your process?
What commands did you run? what tools have you used?

Thanks foro your reply.
Prediction according the method in paper titled " Identification of ribosomal RNA genes in metagenomic fragments"。
And picking 16S by a perl code.
Then input into qiime2 and dereplication, after that clustering into OTU.

Hello @li0604,

There is no requirement that your input sequences be from the V3 or V4 16S regions! You can use all the predicted 16S sequences that you have. That being said, I'm unsure whether dada2 will be applicable to a mixture of fragments of different lengths and from different regions of the 16S gene. You'll have to do some research about that. If that's not possible you can go with the old school clustering approach. You'll also want to be careful when you taxonomically classify and make sure you use a classifier that will work for your unique situation. That will probably be a full-length 16S classifier.

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