what sort of pre-processing should be done prior to picrust2?

I have a question related to the vsearch and the picrust2.

I noticed that in the Overview page of QIIME2 tutorial, there is a sentence:

q2-vsearch implements three different OTU clustering strategies: de novo, closed reference, and open reference. All should be preceded by basic quality-score-based filtering and followed by chimera filtering and aggressive OTU filtering (the treacherous trio, a.k.a. the Bokulich method). :see_no_evil::hear_no_evil::speak_no_evil:

So my question is, which table.qza and rep-seqs.qza should we choose when we want to begin a picrust2 analysis? The two after the culster-features-closed-reference (table-cr-97.qza, rep-seqs-cr-97.qza) or the two finished the chimera filtering(table-cr-97-nonchimeric-wo-borderline.qza, rep-seqs-cr-97-nonchimeric-wo-borderline.qza)?

Not mean to disturb, just feel confused, hope you can help me.

Best wishes,

Mavis

Hello Mavis,

PICRUSt-2 is more flexible than PICRUSt 1, so you can pass in any features (ASVs or OTUs) that you want!

According to the q2 picrust2 tutorial,

This plugin can be integrated with the output files of the default QIIME2 pipelines - i.e. with denoising approaches rather than closed-reference OTU picking.

They are talking about the ASVs made by dada2 from this part of the Moving Pictures Tutorial.

But it looks like you using vsearch to make your features, and picrust2 supports those too! So I would recommend using the same sequences and tables you use for the rest of your analysis.

Consistency is easy to justify. :woman_shrugging:

Colin

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@colinbrislawn Thank you for your reply! But I think I didn’t make the question clear.

My question is: I want to use the .biom and .fna file to do the Picrust analysis, is it necessary for me to use the .biom and .fna file generated by closed reference + chimera filtering, or I can just use the .biom and .fna generated by closed reference (without chimera filtering), or the two kinds of .biom and .fna are both ok?

By the way, I want to use the dada2 pipeline, but I don’t have the .fastq file, I only have .fna file :sob: . I have no choice but to use vsearch.

Thank you again.

Mavis

Hello Mavis,

Now I understand! Closed-ref does not require chimera filtering, so you could just use the closed-ref table.

(Picrust1 needed closed-ref, but picrust2 can work with any method, including vsearch-denovo, dada2, vsearch-open-ref, etc.)

:crying_cat_face: indeed!

Colin

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Got it! And another question occurs to me. :see_no_evil::speak_no_evil::hear_no_evil:

Closed-ref does not require chimera filtering

Can I understand the sentence as following: Closed-ref works according the database, so I don’t need to remove the chimeras from the table and ref-seqs generated by closed-ref, no matter what analysis I want to do in the next step (classification or picrust analysis).

In contrast, de novo and open-ref ask us to do chimeras removing, no matter what analysis I want to do in the next step (classification or picrust analysis), right? :thinking:

Mavis

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Correct! Because closed-ref only reports hits to the database, doing chimera checking is just checking the reference database for chimeras.

Right. De novo OTUs / ASVs have not undergone chimera checking, so it’s your job to do that.

Great work! Let us know if you have any more questions!

Colin

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:partying_face::partying_face::partying_face:

Thank you again for your warm answer! Now I don’t questions any more :100:

And I think your answer will be useful to those who are new to QIIME2 like me, too! What a nice job!

Best wishes.

Mavis

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