Now, we’ll upload some actual microbiome data to explore. To do this, we need to add the data themselves, along with some information telling Qiita about how those data were generated.
Exploring the raw data¶
Click on the 16S menu on the left. Now that you’ve associated sequence files with this prep, you’ll have a “Files network” displayed:
If you see this message:
It means that your files need time to load. Refresh your screen after about 1 minute.
Your collection of FASTQ files for this prep are all represented by a single object in this network, currently called “[user’s_name]”. Click on the object.
Now, you’ll have a series of choices for interacting with this object. You can click “Edit” to rename the object, “Process” to perform analyses, or “Delete” to delete it. In addition, you’ll see a list of the actual files associated with this object.
Scroll to the bottom, and you’ll also see an option to generate a summary of the object.
If you click this button, it will be replaced with a notification that the summary generation has been added to the processing queue.
To check on the status of the processing job, you can click the rightmost icon at the top of the screen:
This will open a dialogue that gives you information about currently running jobs, as well as jobs that failed with some sort of error. Please note, this dialogue keeps the entire history of errors that Qiita encountered for your jobs, so take notice of dates and times in the Hartbeat column.
The summary generation shouldn’t take too long. When it completes, you can click back on the FASTQ object and scroll to the bottom of the page to see a short peek at the data in each of the FASTQ files in the object. These summaries can be useful for troubleshooting.
Now, we’ll process the raw data into something more interesting.
Processing 16S data¶
Scroll back up and click on the “[user’s_name](FASTQ)” artifact, and select “Process”. This will bring you to the workflow network visualization interface. Here, you can add processing steps to your objects.
Below the files network, you will see an option to Choose command. Based on the type of object, this dropdown menu will give a you a list of available processing steps.
For 16S “FASTQ” objects, the only available command is “Split libraries FASTQ”. The converts the raw FASTQ data into the file format used by Qiita for further analysis (you can read more extensively about this file type here).
Select the “Split libraries FASTQ” step. Now, you will be able to select the specific combination of parameters to use for this step in the “Choose parameter set” dropdown menu.
For our files, choose “Multiplexed FASTQ; Golay_12 base pair reverse complement mapping file barcodes with reverse complement barcodes”. The specific parameter values used will be displayed below. For most raw data coming out of the Knight Lab you will use the same setting.
Click “Add Command”.
You’ll see the files network update. In addition to the original grey object, you should now see the processing command (represented in blue) and the object produced from that command (also represented in grey).
You can click on the command to see the parameters used, or on an object to perform additional steps.
Note that the command hasn’t actually been run yet! (We’ll still need to click “Run” at the top.) This allows us to add multiple processing steps to our study and then run them all together.
We’re going to process our sequences files using two different workflows. In the first, we’ll use a conventional reference-based OTU picking strategy to cluster our 16S sequences into OTUs. This approach matches each sequence to a reference database, ignoring sequences that don’t match the reference. In the second, we will use deblur, which uses an algorithm to remove sequence error, allowing us to work with unique sequences instead of clustering into OTUs. Both of these approaches work great with Qiita, because we can compare the observations between studies without having to do any sort of re-clustering!
The closed reference workflow¶
To do closed reference OTU picking, click on the “demultiplexed (Demultiplexed)” object and select the “Pick closed-reference OTUs” command. We will use the “Defaults” parameter set for our data, which are relatively small. For a larger data set, we might want to use the “Defaults - parallel” implementation.
By default, Qiita uses the GreenGenes 16S reference database. You can also choose to use Silva, or the Unite fungal ITS database.
Click “Add Command”, and you will see the network update:
Here you can see the blue “Pick closed-reference OTUs” command added, and that the product of the command is a BIOM-formatted OTU table.
The deblur workflow¶
The deblur workflow is only marginally more complex. Although you can deblur the demultiplexed sequences directly, “deblur” works best when all the sequences are the same length. By trimming to a particular length, we can also ensure our samples will be comparable to other samples already in the database.
Click back on the “demultiplexed (Demultiplexed)” object. This time, select the Trimming operation. Currently, there are seven trimming length options. Let’s choose “Trimming 100”, which trims to the first 100bp, for this run, and click “Add Command”.
Now you can see that we have the same “demultiplexed (Demultiplexed)” object being used for two separate processing steps – closed-reference OTU picking, and trimming.
Now we can click the “Trimmed Demultiplexed (Demultiplexed)” object and add a deblur step. Choose “Deblur” from the “Choose command” dropdown, and “Defaults” for the parameter set.
Add this command to create this workflow:
As you can see, “deblur” produces two BIOM-formatted OTU tables as output. The “deblur reference hit table (BIOM)” contains deblurred sequences that have been filtered to try and exclude things like organellar mitochondrial reads, while “deblur final table (BIOM)” has all the sequences.
Running the workflow¶
Now, we can see the whole set of commands and their output files:
Click “Run” at the top of the screen, and Qiita will start executing all of these jobs. You’ll see a “Workflow submitted” banner at the top of your window.
As noted above, you can follow the process of your commands in the dialogue at the top right of the window.
You can also click on the “Jobs using this data”, and see status updates from the commands running on that object at the bottom of the page:
The full workflow can take time to load depending on the amount of samples and Qiita workload.
Once objects have been generated, you can generate summaries for them just as you did for the original “FASTQ” object.
The summary for the “demultiplexed (Demultiplexed)” object gives you information about the length of sequences in the object:
The summary for a BIOM-format OTU table gives you a histogram of the the number of sequences per sample: