From 91536df1bf4cfb6f656e643d324fe0eb9f68c172 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Thu, 20 Oct 2022 12:13:15 +0100 Subject: [PATCH 1/7] started new section on Harmony wrote intro on "complex" integration tasks and overview of Harmony --- lessons/06_integration.md | 40 ++++++++++++++++++++++++++++++++++++++- 1 file changed, 39 insertions(+), 1 deletion(-) diff --git a/lessons/06_integration.md b/lessons/06_integration.md index 5e4c2d3..1e526e5 100644 --- a/lessons/06_integration.md +++ b/lessons/06_integration.md @@ -45,7 +45,7 @@ Condition-specific clustering of the cells indicates that we need to integrate t **Why is it important the cells of the same cell type cluster together?** -We want to identify _**cell types which are present in all samples/conditions/modalities**_ within our dataset, and therefore would like to observe a representation of cells from both samples/conditions/modalities in every cluster. This will enable more interpretable results downstream (i.e. DE analysis, ligand-receptor analysis. +We want to identify _**cell types which are present in all samples/conditions/modalities**_ within our dataset, and therefore would like to observe a representation of cells from both samples/conditions/modalities in every cluster. This will enable more interpretable results downstream (i.e. DE analysis, ligand-receptor analysis, differential abundance analysis...). In this lesson, we will cover the integration of our samples across conditions, which is adapted from the [Seurat v3 Guided Integration Tutorial](https://satijalab.org/seurat/v3.0/immune_alignment.html). @@ -227,6 +227,44 @@ Since it can take a while to integrate, it's often a good idea to **save the int saveRDS(seurat_integrated, "results/integrated_seurat.rds") ``` + +## **Integrate** or align samples across multiple variables using PCs + +In the section above, we've presented the `Seurat` integration workflow, which uses canonical correlation analysis (CCA) and multiple nearest neighbors (MNN) to find "anchors" and integrate across samples, conditions, modalities, etc. While the `Seurat` integration approach is wildly used and several benchmarking studies support its great performance in many cases, it is important to recognize that **alternative integration algorithms exist and may work better for complex integration tasks** (see [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) for a comprehensive review and comparison of scRNA-seq data integration tools). + +Not all integration algorithms rely on the same methodology, and they do not always provide the same type of corrected output (embeddings, count matrix...). Their performance is also affected by preliminary data processing steps, including which normalization method was used and how highly variable genes (HVGs) were determined. All those considerations are important to keep in mind when selecting a data integration approach for your study. + +**What do we mean by a "complex" integration task?** + +In their benchmarking study, [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) compared the performance of different scRNA-seq integration tools when confronted to different "complex" tasks. The "complexity" may relate to the number of samples (perhaps generated using different protocols) but also to the biological question the study seeks to address (e.g. comparing cell types across tissues, species). In these contexts, you may need to integrate across multiple confounding factors. As usual, you want to select a data integration approach that successfully balances out the following challenges: + +- Correcting for inter-sample variability due to source samples from different donors +- Correcting for variability across protocols/technologies (10X, SMART-Seq2, inDrop...; single-cell vs. single nucleus; variable number of input cells and sequencing depth; different sample preparation steps...) +- Identifying consistent cell types across different tissues (peripheral blood, bone marrow, lung...) and/or different locations (e.g. areas of the brain) +- Keeping apart cell subtypes (or even cell states) that show similar transcriptomes (CD4 naive vs. memory, NK vs NKT) +- Keeping apart cell subtypes that are unique to a tissue/condition +- Conserving the developmental trajectory, if applicable + +Not all tools may perform as well on every task, and complex datasets may require testing several data integration approaches. In doubt, you might want to analyze independently each of the batches you consider to integrate across, in order to define cell identities at this level before integrating and checking that the initially annotated cell types are mixed as expected. + + +### Overview of Harmony + +In this section, we illustrate the use of [`Harmony`](https://portals.broadinstitute.org/harmony/articles/quickstart.html) as a possible alternative to the `Seurat` integration workflow. Compared to other algorithms, `Harmony` presents the following advantages: + +1. Possibility to integrate data across several variables (for example, by experimental batch and by condition) +2. Significant gain in speed and lower memory requirements for integration of large datasets +3. Interoperability with the `Seurat` workflow + +Instead of using CCA, `Harmony` applies a transformation (normalization and scaling) to the principal components (PCs) values (using all available PCs), e.g. as pre-computed with `Seurat`. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters. The diversity of each cluster is then evaluated, to determine whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate upon, as would be expected in a well-integrated dataset. From there, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. The process is iteratively repeated until convergence. If you are interested in a more detailed breakdown of the `Harmony` algorithm, we recommend checking the [advanced vignette](http://htmlpreview.github.io/?https://github.com/immunogenomics/harmony/blob/master/docs/advanced.html) from the package developers. + +**ADD IMAGE** + + +### Implementing Harmony within the Seurat workflow + + + *** *This lesson has been developed by members of the teaching team at the [Harvard Chan Bioinformatics Core (HBC)](http://bioinformatics.sph.harvard.edu/). These are open access materials distributed under the terms of the [Creative Commons Attribution license](https://creativecommons.org/licenses/by/4.0/) (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.* From 1385c6769e58afc7a687fa6270f4db11e53fc6ad Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Thu, 20 Oct 2022 14:27:17 +0100 Subject: [PATCH 2/7] added harmony overview image --- lessons/06_integration.md | 10 ++++++++-- 1 file changed, 8 insertions(+), 2 deletions(-) diff --git a/lessons/06_integration.md b/lessons/06_integration.md index 1e526e5..ebdeb6f 100644 --- a/lessons/06_integration.md +++ b/lessons/06_integration.md @@ -256,9 +256,15 @@ In this section, we illustrate the use of [`Harmony`](https://portals.broadinsti 2. Significant gain in speed and lower memory requirements for integration of large datasets 3. Interoperability with the `Seurat` workflow -Instead of using CCA, `Harmony` applies a transformation (normalization and scaling) to the principal components (PCs) values (using all available PCs), e.g. as pre-computed with `Seurat`. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters. The diversity of each cluster is then evaluated, to determine whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate upon, as would be expected in a well-integrated dataset. From there, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. The process is iteratively repeated until convergence. If you are interested in a more detailed breakdown of the `Harmony` algorithm, we recommend checking the [advanced vignette](http://htmlpreview.github.io/?https://github.com/immunogenomics/harmony/blob/master/docs/advanced.html) from the package developers. +Instead of using CCA, `Harmony` applies a transformation (normalization and scaling) to the principal components (PCs) values (using all available PCs), e.g. as pre-computed with `Seurat`. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define cluster with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate upon, as would be expected in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again in this corrected PC space, and the process is repeated iteratively until convergence. -**ADD IMAGE** +

+ +

+ +_**Image credit:** Korsunsky, I., Millard, N., Fan, J. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 16, 1289–1296 (2019). https://doi.org/10.1038/s41592-019-0619-0_ + +For a more detailed breakdown of the `Harmony` algorithm, we recommend checking [this advanced vignette](http://htmlpreview.github.io/?https://github.com/immunogenomics/harmony/blob/master/docs/advanced.html) from the package developers. ### Implementing Harmony within the Seurat workflow From d92064199de2caa33f739f31d94b84083d63677b Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Thu, 20 Oct 2022 17:52:09 +0100 Subject: [PATCH 3/7] added harmony overview --- img/harmony_overview.jpeg | Bin 0 -> 262893 bytes 1 file changed, 0 insertions(+), 0 deletions(-) create mode 100644 img/harmony_overview.jpeg diff --git a/img/harmony_overview.jpeg b/img/harmony_overview.jpeg new file mode 100644 index 0000000000000000000000000000000000000000..4436d21aa7fb5a0fa38c47c795257bee2900dbb2 GIT binary patch literal 262893 zcmeFZ2V7H6-zR$L(u_z`S`-kFD$-O+M5T#{SU^CCibxY8f)HAwARsj&0)k3tQlvz> 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z?dMZISkU=$R7X&t%Tb^Nb+;kStECrrq=QB(2iN{UvM zPwm?{!<2wW#SaJW%!e5}JifyNJEfLSUB6=_Jhycyx6?y@tiehXGpU<*mAA+$v*1~G zvTJ_S@bOO0fzJDT4G~u65b^x5JXKt8`De$a4TS%jqsq@DUvke!z-31M zoda?ra6JJgc-+Pf6>m??x))i_ogQP78?5wRe|Y=z1H4D{(D?y$VO0n_ z8?&kb;?TQ z@;;Y7kICPHTJ{sad(YIqZL3STmFtLQ^&A;Ic&yiyJ%ya_DqLkrGU}ma4tC-W z77*^|+%;m~!H#%ET%pT=IU6#04SHGQf^}v_f$AZ~(oRBWCjPExI+b|ah;QzzQg)}j z?3j+;GhwxWrXw!L;%-DrU9jq3&fk~>==2Q_Wh&ntqoDHqW7OGn+~MjTnY;&*w>7Qa z{Yq!_8##g#UxQSzi?9#gNia{qUb8$TXhKkPN|J+SC^^ipxHi~vxp}lwi6mk&vS@;3 zLkgBewB21YOktbMF8LUnAhLA57%~)<=lsExxrR7HSEn&4FT3;02=3dw zHs)fGzcUDUKUOuaLIb1r-0a#QZ{1pHI literal 0 HcmV?d00001 From 46e03c2e6fbe68f8c416d2e1ab6a50bcb7da0098 Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Thu, 20 Oct 2022 18:08:29 +0100 Subject: [PATCH 4/7] minor updates --- lessons/06_integration.md | 10 ++++++---- 1 file changed, 6 insertions(+), 4 deletions(-) diff --git a/lessons/06_integration.md b/lessons/06_integration.md index ebdeb6f..c728823 100644 --- a/lessons/06_integration.md +++ b/lessons/06_integration.md @@ -230,13 +230,15 @@ saveRDS(seurat_integrated, "results/integrated_seurat.rds") ## **Integrate** or align samples across multiple variables using PCs -In the section above, we've presented the `Seurat` integration workflow, which uses canonical correlation analysis (CCA) and multiple nearest neighbors (MNN) to find "anchors" and integrate across samples, conditions, modalities, etc. While the `Seurat` integration approach is wildly used and several benchmarking studies support its great performance in many cases, it is important to recognize that **alternative integration algorithms exist and may work better for complex integration tasks** (see [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) for a comprehensive review and comparison of scRNA-seq data integration tools). +In the section above, we've presented the `Seurat` integration workflow, which uses canonical correlation analysis (CCA) and multiple nearest neighbors (MNN) to find "anchors" and integrate across samples, conditions, modalities, etc. While the `Seurat` integration approach is wildly used and several benchmarking studies support its great performance in many cases, it is important to recognize that **alternative integration algorithms exist and may work better for more complex integration tasks** (see [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) for a comprehensive review and comparison of scRNA-seq data integration tools). Not all integration algorithms rely on the same methodology, and they do not always provide the same type of corrected output (embeddings, count matrix...). Their performance is also affected by preliminary data processing steps, including which normalization method was used and how highly variable genes (HVGs) were determined. All those considerations are important to keep in mind when selecting a data integration approach for your study. **What do we mean by a "complex" integration task?** -In their benchmarking study, [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) compared the performance of different scRNA-seq integration tools when confronted to different "complex" tasks. The "complexity" may relate to the number of samples (perhaps generated using different protocols) but also to the biological question the study seeks to address (e.g. comparing cell types across tissues, species). In these contexts, you may need to integrate across multiple confounding factors. As usual, you want to select a data integration approach that successfully balances out the following challenges: +In their benchmarking study, [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) compared the performance of different scRNA-seq integration tools when confronted to different "complex" tasks. The "complexity" of integrating a dataset may relate to the number of samples (perhaps generated using different protocols) but also to the biological question the study seeks to address (e.g. comparing cell types across tissues, species...). In these contexts, you may need to integrate across multiple confounding factors before you can start exploring the biology of your system. + +In these more complex scenarios, you want to select a data integration approach that successfully balances out the following challenges: - Correcting for inter-sample variability due to source samples from different donors - Correcting for variability across protocols/technologies (10X, SMART-Seq2, inDrop...; single-cell vs. single nucleus; variable number of input cells and sequencing depth; different sample preparation steps...) @@ -256,10 +258,10 @@ In this section, we illustrate the use of [`Harmony`](https://portals.broadinsti 2. Significant gain in speed and lower memory requirements for integration of large datasets 3. Interoperability with the `Seurat` workflow -Instead of using CCA, `Harmony` applies a transformation (normalization and scaling) to the principal components (PCs) values (using all available PCs), e.g. as pre-computed with `Seurat`. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define cluster with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate upon, as would be expected in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again in this corrected PC space, and the process is repeated iteratively until convergence. +Instead of using CCA, `Harmony` applies a transformation to the principal components (PCs) values, using all available PCs, e.g. as pre-computed within the `Seurat` workflow. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define cluster with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate on, as would be expected in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again using these corrected PCs, and the process is repeated iteratively until convergence.

- +

_**Image credit:** Korsunsky, I., Millard, N., Fan, J. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 16, 1289–1296 (2019). https://doi.org/10.1038/s41592-019-0619-0_ From 3ec7ad696235739e51e669f9ab5932c54304734d Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Fri, 21 Oct 2022 10:17:45 +0100 Subject: [PATCH 5/7] added code for pre-processing before harmony --- lessons/06_integration.md | 52 +++++++++++++++++++++++++++++++++++++-- 1 file changed, 50 insertions(+), 2 deletions(-) diff --git a/lessons/06_integration.md b/lessons/06_integration.md index c728823..1939d25 100644 --- a/lessons/06_integration.md +++ b/lessons/06_integration.md @@ -252,13 +252,13 @@ Not all tools may perform as well on every task, and complex datasets may requir ### Overview of Harmony -In this section, we illustrate the use of [`Harmony`](https://portals.broadinstitute.org/harmony/articles/quickstart.html) as a possible alternative to the `Seurat` integration workflow. Compared to other algorithms, `Harmony` presents the following advantages: +In this section, we illustrate the use of [`Harmony`](https://portals.broadinstitute.org/harmony/articles/quickstart.html) as a possible alternative to the `Seurat` integration workflow. Compared to other algorithms, `Harmony` notably presents the following advantages ([Korsunsky et al. 2019](https://www.nature.com/articles/s41592-019-0619-0), [Tran et al. 2020](https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1850-9)): 1. Possibility to integrate data across several variables (for example, by experimental batch and by condition) 2. Significant gain in speed and lower memory requirements for integration of large datasets 3. Interoperability with the `Seurat` workflow -Instead of using CCA, `Harmony` applies a transformation to the principal components (PCs) values, using all available PCs, e.g. as pre-computed within the `Seurat` workflow. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define cluster with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate on, as would be expected in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again using these corrected PCs, and the process is repeated iteratively until convergence. +Instead of using CCA, `Harmony` applies a transformation to the principal component (PCs) values, using all available PCs, e.g. as pre-computed within the `Seurat` workflow. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define clusters with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate on, as should be expected in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again using these corrected PCs, and the process is repeated iteratively until convergence.

@@ -271,6 +271,54 @@ For a more detailed breakdown of the `Harmony` algorithm, we recommend checking ### Implementing Harmony within the Seurat workflow +In practice, we can easily use `Harmony` within our `Seurat` workflow. To perform integration, `Harmony` takes as input a *merged* Seurat object, containing data that has been appropriately normalized (i.e. here, normalized using `SCTransform`) and for which highly variable features and PCs are defined. + +There are 2 ways to reach that point: + +1. Merge the *raw* Seurat objects for all samples to integrate; then perform normalization, variable feature selection and PC calculation on this merged object (workflow recommended by `Harmony` developers) +2. Perform (SCT) normalization independently on each sample and find integration features across samples using `Seurat`; then merge these *normalized* Seurat objects, set variable features manually to integration features, and finally calculate PCs on this merged object (workflow best reflecting recommendations for application of `SCTransform`) + +In the first scenario, assuming `raw_seurat_list` is a list of N samples containing raw data that have only undergone QC filtering, we would thus run the following code: + +```r +# Merge raw samples +merged_seurat <- merge(x = raw_seurat_list[[1]], + y = raw_seurat_list[2:length(raw_seurat_list)], + merge.data = TRUE) + +# Perform log-normalization and feature selection, as well as SCT normalization on global object +merged_seurat <- merged_seurat %>% + NormalizeData() %>% + FindVariableFeatures(selection.method = "vst", nfeatures = 2000) %>% + ScaleData() %>% + SCTransform(vars.to.regress = c("mitoRatio")) + +# Calculate PCs using variable features determined by SCTransform (3000 by default) +merged_seurat <- RunPCA(merged_seurat, assay = "SCT", npcs = 50) +``` + +In the second scenario, assuming `norm_seurat_list` is a list of N samples similar to our `split_seurat` object, i.e. containing data that have been normalized as demonstrated in the previous lecture on SCT normalization, we would thus run the following code: + +```r +# Find most variable features across samples to integrate +integ_features <- SelectIntegrationFeatures(object.list = norm_seurat_list, nfeatures = 3000) + +# Merge normalized samples +merged_seurat <- merge(x = raw_seurat_list[[1]], + y = raw_seurat_list[2:length(raw_seurat_list)], + merge.data = TRUE) + +# Manually set variable features of merged Seurat object +VariableFeatures(merged_seurat) <- integ_features + +# Calculate PCs using manually set variable features +merged_seurat <- RunPCA(merged_seurat, assay = "SCT", npcs = 50) +``` + +> _**NOTE:** As mentioned above, there is active discussion within the community regarding which of those 2 approaches to use (see for example [here](https://github.com/immunogenomics/harmony/issues/41) and [here](https://github.com/satijalab/sctransform/issues/55#issuecomment-633843730)). We recommend that you check GitHub forums to make your own opinion and for updates. + + +Regardless of the approach you used, you now have a merged `Seurat` object ready for integration with `Harmony`. *** From 18cfc4952f30c4382e30b716181020b37357151e Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Fri, 21 Oct 2022 11:08:02 +0100 Subject: [PATCH 6/7] added run harmony commands --- lessons/06_integration.md | 31 +++++++++++++++++++++++++++++-- 1 file changed, 29 insertions(+), 2 deletions(-) diff --git a/lessons/06_integration.md b/lessons/06_integration.md index 1939d25..2d03648 100644 --- a/lessons/06_integration.md +++ b/lessons/06_integration.md @@ -307,6 +307,7 @@ integ_features <- SelectIntegrationFeatures(object.list = norm_seurat_list, nfea merged_seurat <- merge(x = raw_seurat_list[[1]], y = raw_seurat_list[2:length(raw_seurat_list)], merge.data = TRUE) +DefaultAssay(merged_seurat) <- "SCT" # Manually set variable features of merged Seurat object VariableFeatures(merged_seurat) <- integ_features @@ -315,11 +316,37 @@ VariableFeatures(merged_seurat) <- integ_features merged_seurat <- RunPCA(merged_seurat, assay = "SCT", npcs = 50) ``` -> _**NOTE:** As mentioned above, there is active discussion within the community regarding which of those 2 approaches to use (see for example [here](https://github.com/immunogenomics/harmony/issues/41) and [here](https://github.com/satijalab/sctransform/issues/55#issuecomment-633843730)). We recommend that you check GitHub forums to make your own opinion and for updates. +> _**NOTE:** As mentioned above, there is active discussion within the community regarding which of those 2 approaches to use (see for example [here](https://github.com/immunogenomics/harmony/issues/41) and [here](https://github.com/satijalab/sctransform/issues/55#issuecomment-633843730)). We recommend that you check GitHub forums to make your own opinion and for updates._ -Regardless of the approach you used, you now have a merged `Seurat` object ready for integration with `Harmony`. +Regardless of the approach, we now have a merged Seurat object containing normalized data for all the samples we need to integrate, as well as defined variable features and PCs. +One last thing we need to do before running `Harmony` is to **make sure that the metadata of our Seurat object contains one (or several) variable(s) describing the factor(s) we want to integrate on** (e.g. one variable for `sample_id`, one variable for `experiment_date`). + +We're then ready to run `Harmony`! + +```r +harmonized_seurat <- RunHarmony(merged_seurat, + group.by.vars = c("sample_id", "experiment_date"), + reduction = "pca", assay.use = "SCT", reduction.save = "harmony") +``` + +The line of code above adds a new reduction of 50 "harmony components" (~ corrected PCs) to our Seurat object, stored in `harmonized_seurat@reductions$harmony`. + +To make sure our `Harmony` integration is reflected in the data visualization, we still need to generate a UMAP derived from these harmony embeddings instead of PCs: + +```r +harmonized_seurat <- RunUMAP(harmonized_seurat, reduction = "harmony", assay = "SCT", dims = 1:40) +``` + +Finally, when running the clustering analysis later on (see next lecture for details), we will also need to set the reduction to use as "harmony" (instead of "pca" by default). + +```r +harmonized_seurat <- FindNeighbors(object = harmonized_seurat, reduction = "harmony") +harmonized_seurat <- FindClusters(harmonized_seurat, resolution = c(0.2, 0.4, 0.6, 0.8, 1.0, 1.2)) +``` + +> _**NOTE**: You can specify however many variables to integrate on using the `group.by.vars` parameter, although we would recommend keeping these to the minimum necessary for your study._ *** From 7b83c145eec7c3973b99371bbeb930844d088a6e Mon Sep 17 00:00:00 2001 From: =?UTF-8?q?Am=C3=A9lie=20Jul=C3=A9?= Date: Fri, 21 Oct 2022 11:19:45 +0100 Subject: [PATCH 7/7] minor edits --- lessons/06_integration.md | 14 ++++++++------ 1 file changed, 8 insertions(+), 6 deletions(-) diff --git a/lessons/06_integration.md b/lessons/06_integration.md index 2d03648..14bf49e 100644 --- a/lessons/06_integration.md +++ b/lessons/06_integration.md @@ -230,13 +230,13 @@ saveRDS(seurat_integrated, "results/integrated_seurat.rds") ## **Integrate** or align samples across multiple variables using PCs -In the section above, we've presented the `Seurat` integration workflow, which uses canonical correlation analysis (CCA) and multiple nearest neighbors (MNN) to find "anchors" and integrate across samples, conditions, modalities, etc. While the `Seurat` integration approach is wildly used and several benchmarking studies support its great performance in many cases, it is important to recognize that **alternative integration algorithms exist and may work better for more complex integration tasks** (see [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) for a comprehensive review and comparison of scRNA-seq data integration tools). +In the section above, we've presented the `Seurat` integration workflow, which uses canonical correlation analysis (CCA) and multiple nearest neighbors (MNN) to find "anchors" and integrate across samples, conditions, modalities, etc. While the `Seurat` integration approach is wildly used and several benchmarking studies support its great performance in many cases, it is important to recognize that **alternative integration algorithms exist and may work better for more complex integration tasks** (see [Luecken et al. (2022)](https://doi.org/10.1038/s41592-021-01336-8) for a comprehensive review). Not all integration algorithms rely on the same methodology, and they do not always provide the same type of corrected output (embeddings, count matrix...). Their performance is also affected by preliminary data processing steps, including which normalization method was used and how highly variable genes (HVGs) were determined. All those considerations are important to keep in mind when selecting a data integration approach for your study. **What do we mean by a "complex" integration task?** -In their benchmarking study, [Luecken et al (2022)](https://doi.org/10.1038/s41592-021-01336-8) compared the performance of different scRNA-seq integration tools when confronted to different "complex" tasks. The "complexity" of integrating a dataset may relate to the number of samples (perhaps generated using different protocols) but also to the biological question the study seeks to address (e.g. comparing cell types across tissues, species...). In these contexts, you may need to integrate across multiple confounding factors before you can start exploring the biology of your system. +In their benchmarking study, [Luecken et al. (2022)](https://doi.org/10.1038/s41592-021-01336-8) compared the performance of different scRNA-seq integration tools when confronted to different "complex" tasks. The "complexity" of integrating a dataset may relate to the number of samples (perhaps generated using different protocols) but also to the biological question the study seeks to address (e.g. comparing cell types across tissues, species...). In these contexts, you may need to integrate across multiple confounding factors before you can start exploring the biology of your system. In these more complex scenarios, you want to select a data integration approach that successfully balances out the following challenges: @@ -258,7 +258,7 @@ In this section, we illustrate the use of [`Harmony`](https://portals.broadinsti 2. Significant gain in speed and lower memory requirements for integration of large datasets 3. Interoperability with the `Seurat` workflow -Instead of using CCA, `Harmony` applies a transformation to the principal component (PCs) values, using all available PCs, e.g. as pre-computed within the `Seurat` workflow. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define clusters with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate on, as should be expected in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again using these corrected PCs, and the process is repeated iteratively until convergence. +Instead of using CCA, `Harmony` applies a transformation to the principal component (PCs) values, using all available PCs, e.g. as pre-computed within the `Seurat` workflow. In this space of transformed PCs, `Harmony` uses k-means clustering to delineate clusters, seeking to define clusters with maximum "diversity". The diversity of each cluster reflects whether it contains balanced amounts of cells from each of the batches (donor, condition, tissue, technolgy...) we seek to integrate on, as should be observed in a well-integrated dataset. After defining diverse clusters, `Harmony` determines how much a cell's batch identity impacts on its PC coordinates, and applies a correction to "shift" the cell towards the centroid of the cluster it belongs to. Cells are projected again using these corrected PCs, and the process is repeated iteratively until convergence.

@@ -304,8 +304,8 @@ In the second scenario, assuming `norm_seurat_list` is a list of N samples simil integ_features <- SelectIntegrationFeatures(object.list = norm_seurat_list, nfeatures = 3000) # Merge normalized samples -merged_seurat <- merge(x = raw_seurat_list[[1]], - y = raw_seurat_list[2:length(raw_seurat_list)], +merged_seurat <- merge(x = norm_seurat_list[[1]], + y = norm_seurat_list[2:length(raw_seurat_list)], merge.data = TRUE) DefaultAssay(merged_seurat) <- "SCT" @@ -330,6 +330,7 @@ harmonized_seurat <- RunHarmony(merged_seurat, group.by.vars = c("sample_id", "experiment_date"), reduction = "pca", assay.use = "SCT", reduction.save = "harmony") ``` +> _**NOTE**: You can specify however many variables to integrate on using the `group.by.vars` parameter, although we would recommend keeping these to the minimum necessary for your study._ The line of code above adds a new reduction of 50 "harmony components" (~ corrected PCs) to our Seurat object, stored in `harmonized_seurat@reductions$harmony`. @@ -346,7 +347,8 @@ harmonized_seurat <- FindNeighbors(object = harmonized_seurat, reduction = "harm harmonized_seurat <- FindClusters(harmonized_seurat, resolution = c(0.2, 0.4, 0.6, 0.8, 1.0, 1.2)) ``` -> _**NOTE**: You can specify however many variables to integrate on using the `group.by.vars` parameter, although we would recommend keeping these to the minimum necessary for your study._ +The rest of the `Seurat` workflow and downstream analyses after integration using `Harmony` can then proceed without further amendments. + ***