Assessing metagenome contributions using output from Qiime2 PICRUSt2 plugin

[Kirk B]

Hi there, I have successfully navigated the PICRUSt2 pipeline using the new plugin in Qiime2. Now I would like to input the predicted metagenome into HUMAnN2 and stratify the metagenome contributions by ASV/taxon. Metagenome_contributions.py in PICRUSt1 provided taxon contributions to metagenomes, however, I am unclear how I can get an equivalent file in the q2 PICRUST2 plugin.  Any help would be much appreciated.




Best regards,

Kirk


[Gavin Douglas]

Hey Kirk,

I haven’t implemented stratified output with the QIIME2 plugin yet. You’ll need to directly run the Python scripts to get stratified tables with the current version. Note that you need to specify an option to get stratified output (e.g. “--strat_out” with metagenome_prediction.py) and this can result in much higher memory usage and longer running times.

Best,

Gavin

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[Kirk B]

Hi Gavin - thanks for your quick reply and great work with Microbiome Helper and Picrust2 development.  I will try the full PICRUSt2 pipeline with the stratify options.  Is it feasible to run this using MacOS/X (on an iMac) (worried about the EPA-NG and GAPPA installations) , or do you recommend using the VM Linux Ubuntu?

- Thank you, Kirk

[Gavin Douglas]

You will likely run into issues installing EPA-NG on Mac OS X, but you could simply export the tree you created with qiime fragment-insertion and input that into the next step of the PICRUSt2 pipeline to run it on your Mac. Note that if you do use the MH VBox image that I have made updates to PICRUSt2 since the last release of the image so you should re-install the latest version of PICRUSt2.

Best,

Gavin

[Kirk B]

Great - thank you Gavin - I will try those suggestions.

Best, Kirk

[Jonathan Gaiero]

Hi Gavin,

We were able to run the picrust2 qiime2 plugin (VM with qiime2 image) and the outputs look pretty good. Is there a way to see the NSTI values for each sample or ASV? 

It seems as though this data pops out from the full pipeline using python. Should we be looking into using the full pipeline command in the plugin instead of the 'custom-pipeline' as in the tutorial?

Another question-  overall, would the results differ much between the qiime2 picrust2 plugin and the full pipeline using python?

Thanks for your help!

Jon

[Gavin Douglas]

Hi there,

I haven’t implemented those features into the QIIME2 plugin yet. You can get the NSTI values by ASV and weighted NSTI values per sample with the main PICRUSt2 workflow: https://github.com/picrust/picrust2/wiki/Workflow

The results shouldn’t differ much - they would only differ a little based on the different placement procedures, but this should be very minor.

Gavin

[Jonathan Gaiero]

OK. Thank you for the response!

[Aqleem Abbas]

Dear Sir, 

Can You provide me step by step protocol of exporting my feature table and reference sequences to Picrust. You have told that you have become succesfull.

Thanks 

[Ravinder Nagpal]

Hi Gavin, 

We occasionally do 16S sequencing using Earth Microbiome Project protocol and run QIIME on base-space sequence hub apps i.e. QIIME preprocessing and QIIME visualizations. Can we expect these basespace apps to be updated to latest QIIME version 2? That would be so facilitating for numerous users like us and will be highly appreciated.

Thanks

Rav

  

[Gavin Douglas]

Hi Rav,

This is the google group for PICRUSt, which is an independent tool from QIIME2. It’s a little confusing since there is a PICRUSt2 plugin in QIIME2 though. QIIME2 is a framework for using many different bioinformatic tools and there is a growing number of plugins from independent developers. You can find more info about QIIME2 here: https://qiime2.org/

In the future please create a new thread rather than adding to an unrelated thread if you have any questions.

Thanks,

Gavin

[Gavin Douglas]

Hi Aqleem,

I’ll need more details to help you out. Could you please create a new thread on this google group and describe what form your data is currently in and what commands you’re trying to run? It’s best not to respond to unrelated threads as this creates confusion. In the future please create a new thread if you have a question. I believe you have a feature table and representative sequences output by QIIME2. If so, I have previously linked you to this tutorial: https://github.com/picrust/picrust2/wiki/q2-picrust2-Tutorial. If you’re confused about anything in particular on that or another page I’m happy to help.

Best,

Gavin

[Aqleem Abbas]

Dear Sir,

Thank you sharing the tutorial again. I have some basic questions. I have at the moment the feature table and feature sequences from Qiime 2. How to import these two into picrust for analysis? I have read the tutorial that you have sent to me however I could not understand some commands e.g. The required inputs are --i-table and --i-tree, which need to correspond to QIIME2 artifacts of types FeatureTable[Frequency] and Phylogeny[Rooted], respectively . Could you please elaborate? I want to become expert on picrust.

Thanks

Aqleem Abbas

[Ravinder Nagpal]

Alright, Gavin! Thx, Rav.

[Gavin Douglas]

The FeatureTable[Frequency] object refers to your input table in QZA format. The “Phylogeny[Rooted]” object refers to your feature sequences placed into the reference tree using the “qiime fragment-insertion sepp” command described in the tutorial.

You input your feature table and the tree with your placed feature sequences into the "qiime picrust2 custom-tree-pipeline” command. You don’t need to export your QIIME2 files to a different format to use PICRUSt2 - you can use the QZA files if you use the q2-picrust2 plugin.

Again in the future please create a new thread if you have a question. To do that you would go to https://groups.google.com/forum/#!forum/picrust-users and click “New Topic”.

Best,

Gavin

[Aqleem Abbas]

Dear Sir,

ok. Thanks

Aqleem Abbas

[Tulsi Joishy]

Hello everyone,

I am trying to do PICRUSt 2 and m very new to it and getting error,  m confused with 

1. rep-seqs.qza- should be aligned one?

2. reference alignment- is it the classifier file with which i perform qiime2 analysis.

3. rooted tree- should it be the output of phylogeny command but of aligned re-seqs.qza or not aligned ??

i typed this command and got this error:

(qiime2-2018.11) [qiime2@localhost PICRUSt2]$ qiime fragment-insertion sepp --i-representative-sequences rep-seqs.qza \

>                               --p-threads 1 --i-reference-alignment /home/qiime2/classifier/gg-13-8-v3-v4-classifier.qza \

>                               --i-reference-phylogeny 2-rooted-tree.qza \

>                               --output-dir Picrust2_output

Plugin error from fragment-insertion:

  Parameter 'reference_alignment' received an argument of type TaxonomicClassifier. An argument of subtype FeatureData[AlignedSequence] is required.

Debug info has been saved to /tmp/qiime2-q2cli-err-n44sms7c.log

Please help.

[Aqleem Abbas]

Dear Sir,

I could not install picrust2 plugin in the recent version of Qiime 2. I followed two steps in tutorial but the third step has given an error.

Thanks

Aqleem Abbas

--

[Anwesh Maile]

You need to download the reference files given in the q2-picrust2 tutorial and use them as reference alignment and reference phylogeny...

wget http://kronos.pharmacology.dal.ca/public_files/tutorial_datasets/picrust2_tutorial_files/reference.fna.qza

wget http://kronos.pharmacology.dal.ca/public_files/tutorial_datasets/picrust2_tutorial_files/reference.tre.qza

[Tulsi Joishy]

Thank you Anwesh!!...I could do that but now having trouble in PICRUST2 installation.

[spark]

Hi Tulsi

Did you get it to work? I used the qiime2 plugin from 

I did 

/workspace/hraegb/singularity/qiime-2019.7-picrust2.sif picrust2 full-pipeline \

--i-table $FILTER/table-dada2.qza \

--i-seq $FILTER/rep-seqs-dada2.qza \

--output-dir $WORKING/$PICR2/pic \

--o-ko-metagenome $PICR2/KO \

--o-ec-metagenome $PICR2/EC \

--o-pathway-abundance $PICR2/PA \

--p-threads 1 \

--p-hsp-method mp \

--p-max-nsti 2 \

--verbose

I got it to work. So I didn't require the rooted tree.

thanks

spark