Special thanks to Anonymous Commenter "B" and The Self-Taught Author as well as Richard Sprague for the links, guidance and patience to work your way through all these metagenomic tools. I feel we learn a bit more every day, even if it's not what we hoped to learn!
I wrote these instructions out as I went through analyzing some of my own gut results. Let me know how they work if you try.
- First, go to the Euro Nucleotide Archive biosample page and enter your 9 digit AmGut kit #, example 000016449, and search by 'samples' from dropdown (default).
- One of the results you get should say in the description "American Gut Project Stool sample"
- Click the link under 'Accession' and make sure this is your sample.
- Scroll to bottom and click the link next to "databases" (ex: ERS577362)
- Scroll down and look for a column labeled "Fastq files (ftp)", click on File 1.
- When prompted, save this file to your desktop or somewhere handy, change the name from the default (ERR667803.fastq.gz) to something more appropriate (AmGut2.fastq.gz).
- You are done with Step 1
- Log into MG-Rast. Create an account if needed.
- Click the Green UP arrow in upper right hand corner box to upload the file you just made in step 1.
- Skip their step 1, go to step 2, find the fastq.gz file you just created and upload it. Do not generate a webkey, not needed. Don't do the Md-5 check, just close it when prompted.
- Go to step 3, manage Inbox. Highlight your new file, then select 'unpack selected' (takes a few minutes to decompress).
- Click 'update inbox', the fastq.gz file changes to .fasta. Highlight the new file and see that there are no errors.
- Click Step 1 under Data Submission, click the "I do not want to supply metadata" box, and select.
- Step 2, create a new project with unique name, or use existing.
- Step 3, You should see your file here, check the box and then 'select'.
- Step 4, leave all as default, click 'select'.
- Step 5, click the very top box, for highest priority (otherwise it will take days!), Submit the job, and make a note of the files in the popup box for future reference. Click OK, then you'll get a "Successful Job" popup with another number, write that down, too!
- To check progress of new jobs, click on the Globe Icon in upper right corner
- In the "Browse Metagenomes" section, you'll see a listing of your jobs and files. "In progress" shows how many you are waiting on.
- Click the little linked number 'available for analysis', and you'll get a table with all of your files.
- Click the link under 'name' column and you'll get a good analysis of your file, but you can also analyze the data using Step 4 procedures.
- Click the 'barchart' icon in top right corner.
- Under 'data selection', expand the 'metagenomes' This will give a list of all that you have put into MG-rast.
- Select a Genome from the dropdown
- Use the arrows to slide it over to the box on the right. Once you get the hang, you can select 2 samples and compare them.
- Change 'annotation sources' from M5NR to GreenGenes. Green Genes is what AmGut uses. It's a master list of microbes. Remember, they can't see what microbe they are looking at, they can only compare it to a known microbe in a 16srRNA library! These sources are the different libraries.
- Leave all other parameters alone.
- Now, later you can play, but choose 'Table' and 'generate'
- Group table by Species, and hit change.
- You'll have hundreds of line items, 15 or so per page. Scroll through and you'll see what was in the sample.