Does anyone know how to use PICRUST with the data obtained from Kraken?

[Ana Benavides]

Does anyone know how to use PICRUST with the data obtained from Kraken?

[Robyn Wright]

Hi Ana,

 

Can you give me some details about what kind of data you’re working with? Kraken is most commonly used with metagenomic data, in which case you don’t need to predict functions (what PICRUSt is used for), because you can just annotate the functions within your data. In this case, I would recommend following our labs Metagenome workflow here.

 

If your data is 16S amplicon sequencing, it doesn’t really matter which method you have used for taxonomic classification as what you need for PICRUSt is your ASV/OTU sequences and a table with the abundance of these sequences across your samples. You can see descriptions of these files here.

 

Thanks,

Robyn

 

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[Ana Benavides]

Hi Robyn 

Thanks for your response. 

They are 16S and ITS metabarcoding data. I use Kraken for assignment, but I don't know how to obtain this study_seqs.fna file. (ASV/OTU sequences)

Ana,

[Robyn Wright]

Hi Ana,

 

I’m not familiar with any workflows that use Kraken for taxonomic assignment, but usually in amplicon analysis (e.g. our lab workflow here) you would:

• Inspect the read quality
• Trim the primers from the reads
• Filter low quality reads
• Denoise the reads into ASVs/cluster into OTUs (and also join the forward and reverse reads in this step) – from this step you’ll typically have a fasta file of denoised/representative sequences, as well as a feature table that has the abundance of the ASVs/OTUs across samples
• Assign taxonomy
• Filter rare, contaminant and unclassified ASVs

 

Usually it would be the sequence file that you have at the end, where the rare, contaminant and unclassified ASVs have been removed, that you would use for input into PICRUSt2. If you have details of your workflow (all of the commands that you run) then maybe I can advise better.

 

Robyn

 

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[Ana Benavides]

Hi Robyn,

Thanks. I use Kraken2 in my pipeline with the following command:

kraken2 --use-names --db $DBNAME --threads 24 --report name.report

Ana

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[Robyn Wright]

Hi Ana, 

The command you've shown doesn't tell me which files you use for running kraken2, or what they look like. Before running PICRUSt2, you need to identify ASVs/OTUs and get their abundance in your samples. You can see examples of these files in the tutorial, but you need these before you can run PICRUSt2. There are numerous tutorials available for processing your amplicon sequencing data (e.g. our Microbiome Helper tutorial), but you need these before proceeding to analysis with PICRUSt2.

Best wishes,

Robyn