QA/QC

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& Data Processing

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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 (

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e.g. NegProbes).

• Click on  Filtering > Filter features from the menu on the right.
• Select Metadata and set the task settings as follows, then click
• Click Finish:

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In the Analyses tab

• Click on the resulting filtered counts node and select 
• 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-depended dependent and will change based on your dataset). Then click Image Removed and
• Click FilterImage Added include
• Click Apply observation filter to the filtered counts node:

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Click on the node generated by the filtering task

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in the Analyses tab.

• Click Filtering > Filter features. Apply a noise reduction filter:

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We can now

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normalize our filtered data.

• Click Normalization and scaling > NormalisationNormalization. Use the recommended settings by clicking :

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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):

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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

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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:

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