Shotgun metagenomics is an alternative approach to amplicon sequencing that provides information at the genomic level, rather than for single loci such as 16S, 18S, and the fungal ITS region. In addition to providing finer resolution with respect to taxonomic classification, metagenomics provides functional data that allows for more comprehensive analyses of microbial communities. We are currently working to make available additional metagenomic tools in Qiita. Below we describe how to use Shogun to produce taxonomic profiles for shotgun metagenomic data, and then move to common downstream analyses of alpha- and beta-diversity.

Processing Shotgun Metagenomics Data

We will start by creating a new study with shotgun metagenomic data for 23 human fecal samples subsetted from the American Gut Project. As above for amplicon data, go to the study drop-down menu and select ‘Create Study’. Complete the necessary fields similar as shown below.

_images/shotgun-process-create-study.png

Once the study has been created, click on the study name in the green banner to go to the study page. Click on ‘Upload Files’, and upload the contents of the shotgun data.

_images/shotgun-process-upload.png

Once the files are uploaded, click on ‘Sample Information’ on the left side of the screen and select the sample information file ‘CMI_shotgun-metagenomics_tutorial_sample_info.txt’ and click ‘Create’.

_images/shotgun-process-sample-info.png

Once complete, click ‘Add New Preparation’. Give the preparation a name of your choosing, select the prep info file, ‘CMI_shotgun-metagenomics_tutorial_prep_info’, and select ‘Metagenomic’ in both of the drop-down menus. Then click ‘Create New Preparation’.

_images/shotgun-process-prep-info.png

This will create a new preparation under ‘Data Types’. Click on the new preparation and then the prep ID text. This will open up options for which you should select ‘per_sample_FASTQ - None’, and give the files a name. Then click ‘Add Files’.

_images/shotgun-process-add-files.png

Once your sequence files are added, you should see a processing network with an artifact representing the sequence data.

_images/shotgun-process-network.png

We will now process the sequence data using Shogun to create taxa-abundance tables at three hierarchical levels: phylum, family, and genus. Shogun is a shallow shotgun metagenomics analysis tool that we will use for taxonomic classification of sequences using a reference database. In Qiita, Shogun uses either utree or bowtie2 to align to references.

Running Shogun on human-filtered per-sample FASTQ data

All shotgun metagenomic data is filtered of human reads prior to being uploaded to Qiita. If you have additional, non-human host reads that you would like to filter from your data, you can see if they are available under the command, ‘QC_Filter’. To proceed with processing the data using Shogun, click on the per-sample FASTQ artifact named ‘Shotgun data’ and click ‘Process’.

_images/shotgun-process-shogun1.png

From the dropdown menu, select ‘Shogun’.

_images/shotgun-process-shogun2.png

Change the ‘Parameter set’ to ‘rep82_utree’ and click ‘Add Command’.

_images/shotgun-process-shogun3.png

You should see the command added to the processing network:

_images/shotgun-process-shogun4.png

Click ‘Run’ to run the command.

_images/shotgun-process-shogun5.png

Once the command has completed, click on the ‘Taxonomic Predictions - species’ artifact.

_images/shotgun-process-shogun6.png

Examine the table summary to see the number of samples that were retained, and the number of features that were assembled.

_images/shotgun-process-taxa-species.png

In the next section we will begin an analysis starting with this feature-table.