Analyze Your 16S rRNA Data Using RDP-Classifier

Step #1: Convert Files from FASTQ to FASTA

There are lots of ways to do this, but I would recommend using Galaxy if you don't have any programming experience:

Go to the Galaxy website: https://usegalaxy.org/

If you are an academic researcher, your institution might have a local mirror (which should be faster).  However, the link above will work for everybody.

Upload your data using "Get Data" --> "Upload File" (the functions are available on the left-hand side of the screen).  You can set the file type to "fastq", but you probably don't need to.  Updates will appear on the right-hand side of the screen, so you know when each step is complete (the box for the corresponding step will turn green).

Go to "NGS: QC and manipulation" --> "FASTQ Groomer" (should be under "ILLUMINA DATA" in grey font).  Leave all the default settings and click "Execute".  This is technically necessary because of a formatting issue.

Go to "Convert Formats" --> "FASTQ to FASTA".  Once this step is complete, click the appropriate green box on the right-hand side.  Once the box becomes larger (allowing you to see the first few lines of the file), click the purple floppy disk icon to download the FASTA file.  I would recommend renaming the FASTA file after it is downloaded, so it is easier to keep track of.

Step #2: Create an RDP Account

You can sign up using this link: https://rdp.cme.msu.edu/user/createAcct.spr

An account will be created automatically.  You will receive an e-mail with a username and password (you will be asked to change your password the first time you sign in).  Technically, you don't need an account to run the classifier.  However, I think it may be helpful if you want to play around with some other tools.

Step #3: Sign-In and Run RDP-Classifier

Using the link provided by the registration e-mail, sign into myRDP.

Now, go to this link: https://rdp.cme.msu.edu/classifier/classifier.jsp

There will be an option to "Choose a file (unaligned format) to upload:".  Use the browser to select the FASTA (not FASTQ) file that you downloaded from Galaxy.  Next, click "Submit".

The classifier is very fast (you should get your results in a few minutes).  The result page is somewhat hard to parse, but everything is clickable to learn more.  The number of reads is shown in parentheses.

It really helps to remember biological classifications when interpreting these results.  Here is a quick cheat sheet:

phylum > class > order (> suborder) > family > genus

Unfortunately, the classifier won't provide species-specific information.

You can also download the results in a text file.  If you do this, you can use a tool like Notepad++ to search for keywords (like phylum, genus, etc.), but I think the results are a little easier to view on the webpage.