QA/QC & Data Processing
Once the data has been imported in the project we can start pre-processing the data:
We will first remove all non-expression features in the data (e.g. NegProbes).
- Click on Filtering > Filter features from the menu on the right
- Select Metadata and set the task settings as follows
- Click Finish
In the Analyses tab
- Click on the resulting filtered counts node
- Select QA/QC > Single cell QA/QC from toolbox, once the task has completed we can open the report by double-clicking the node:
We will remove the cells with low counts and number of detected features.
- Click on Select & Filter and set lower threshold to 50 for both (remember that this is data-dependent and will change based on your dataset)
- Click Filter include
- Click Apply observation filter to the filtered counts node:
Click on the node generated by the filtering task in the Analyses tab.
- Click Filtering > Filter features. Apply a noise reduction filter:
We can now normalize our filtered data.
- Click Normalization and scaling > Normalization. Use the recommended settings by clicking :
Data Exploration
Now that we have filtered low quality cells and normalized our data, we can start clustering to identify cell populations.
- Click on the normalized data node
- From the menu on the right select Exploratory analysis > PCA. We are going to use the top 2000 features by variance and calculate the first 50 principal components (PCs):
Once the PCA has run, click on the PCA result node in the Analyses tab.
- Select Exploratory analysis > UMAP from the toolbox. Set the UMAP parameters as follows:
- Top 20 PCs
- Local neighborhood size 60
- Minimal distance 0.20
While the UMAP is running we can also queue a clustering task. Select Exploratory analysis > Graph-based clustering.
- We are going to use the Leiden algorithm to cluster our data (make sure to select the radio button for it)
- Set the number of PCs to 10
- In the advanced settings, set the resolution parameter to 8e-5 and click Apply:
Additional Assistance
If you need additional assistance, please visit our support page to submit a help ticket or find phone numbers for regional support.
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