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Update method summary
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agdenadel committed Feb 28, 2024
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Expand Up @@ -56,7 +56,7 @@ The `callback` algorithm consists of three simple steps:

1. First, we generate synthetic null variables, formally called knockoff features, where we augment the single-cell data being analyzed with "fake" genes that are known not to contribute to any unique cell type.
2. Second, we perform both preprocessing and clustering on this augmented dataset.
3. Third, we calibrate the number of inferred clusters by using a hypothesis testing strategy with a data-dependent threshold to determine if there is a statistically significant difference between groups and if re-clustering should occur.
3. Third, we calibrate the number of inferred clusters by using a hypothesis testing strategy with a data-dependent threshold to determine if there is a statistically significant difference between groups. If any pair of groups does not have statistically significant differences then re-clustering occurs.

The synthetic knockoff genes act as negative control variables; they go through the same analytic steps as the real data and are presented with the same opportunity to be identified as marker genes. The `callback` algorithm uses the guiding principle that well-calibrated clusters (i.e., those representing real groups) should have significantly differentially expressed genes after correcting for multiple hypothesis tests, while over-clustered groups will not. We use this rule to iteratively re-cluster cells until the inferred clusters are well-calibrated and the observed differences in expression between groups are not due to the effects of double-dipping.

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