Answers.Body: <p>See <a href="https://www.ncbi.nlm.nih.gov/pubmed/29608177" rel="nofollow noreferrer">this paper from the Marioni group</a>, where they propose a method for correcting batch effects between single cell sequencing experiments when each experiment contains different sub-populations:</p>
  
  <blockquote>
    <p>Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are
    produced in different laboratories and at different times contain
    batch effects that may compromise the integration and interpretation
    of the data. Existing scRNA-seq analysis methods incorrectly assume
    that the composition of cell populations is either known or identical
    across batches. We present a strategy for batch correction based on
    the detection of mutual nearest neighbors (MNNs) in the
    high-dimensional expression space. Our approach does not rely on
    predefined or equal population compositions across batches; instead,
    it requires only that a subset of the population be shared between
    batches. We demonstrate the superiority of our approach compared with
    existing methods by using both simulated and real scRNA-seq data sets.
    Using multiple droplet-based scRNA-seq data sets, we demonstrate that
    our MNN batch-effect-correction method can be scaled to large numbers
    of cells</p>
  </blockquote>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-06-07T12:38:22.383Z
Answers.LastActivityDate: 2018-06-07T12:38:22.383Z
Answers.UserDisplayName: Ian Sudbery
Answers.Body: <p>Have you tried seurat3? Here is the reference:
  <a href="https://satijalab.org/seurat/v3.0/pancreas_integration_label_transfer.html" rel="nofollow noreferrer">https://satijalab.org/seurat/v3.0/pancreas_integration_label_transfer.html</a></p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-06-27T03:11:00.333Z
Answers.LastActivityDate: 2019-06-27T03:11:00.333Z
Answers.UserDisplayName: Zhi-Ping Feng
Answers.Body: <p>It’s right there on the <a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger" rel="nofollow noreferrer">cellranger manual</a>:</p>
  
  <pre><code>#aggregate results of counts for separate samples
  cellranger aggr
  #analyse the combined results
  cellranger reanalyze
  </code></pre>
  
  <p>Note: I’m not sure this applies to the above case where batch effects could be an issue. However, this is how the analysis is performed for 10X data from multiple runs in principle.</p>
  
  <p><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/aggregate" rel="nofollow noreferrer">cellranger aggr</a></p>
  
  <p><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/reanalyze" rel="nofollow noreferrer">cellranger reanalyze</a></p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-03-04T21:03:33.057Z
Answers.Text: Adding to this answer months later: it does not work with different designs, it just runs depth normalization but it can be severely affected by batch effects.
Answers.UserDisplayName: gc5
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-03-05T00:42:29.917Z
Answers.Text: Yes batch effects must be considered. Since this question was specifically about combining datasets, that is what was described here. This is the first step to adjust for batch effects, there are several methods to do this on an aggregate dataset and there are separate questions on this already (these assume some common cells between samples or it will overfit). I recommend not to use downsampling but aggregate raw. Depth normalisation does not remove batch effects or background noise in each sample.
Answers.UserDisplayName: Tom Kelly ケリー・トム
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-03-05T15:58:30.367Z
Answers.Text: I agree. As you said, methods like ComBat are not suitable for experiments with different design because they require a balanced design (some common cells between samples). Thanks.
Answers.UserDisplayName: gc5
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-03-05T23:19:34.800Z
Answers.Text: The 3.0 release of the Seurat R package (currently in alpha) will have extensive support for combining data from different experiments.
Answers.UserDisplayName: Tom Kelly ケリー・トム
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-06-06T21:47:11.513Z
Answers.LastActivityDate: 2018-06-06T21:47:11.513Z
Answers.UserDisplayName: Tom Kelly ケリー・トム
Body: <p>I have 4 different single-cell RNAseq experiments, each one representing a different sample of cell type population. I'd like to merge them to a single dataset. However, different cell types are enriched in each sample, and different samples can represent also different conditions (control and disease).</p>
  
  <p>If I run a PCA on the merged dataset (without any processing of data) I find that the different sample's cells cluster separately. This could be due to biological and technical (i.e. batch effects) variation.</p>
  
  <p>Is it possible to merge these different samples while retaining a good amount of information or should they be analyzed separately?</p>
  
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2018-02-19T21:21:15.790Z
Comments.Text: Is there some samples (controls perhaps) in both experiments? What is your goal by merging them: compare a cell line of experiment A with a cell line in experiment B?
Comments.UserDisplayName: llrs
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2018-02-19T21:26:37.137Z
Comments.Text: No controls unfortunately. My goal by merging them is basically to find different sub-populations in the data and markers by exploiting a larger dataset. This data was presented to me after sequencing, and I think that it is only possible to use each experiment separately, but I'd like to be proven wrong - at least in some specific analysis.
Comments.UserDisplayName: gc5
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2018-02-19T21:28:56.663Z
Comments.Text: I'm not familiar enough with the methods for scRNA, sorry. But why do you think that increasing the number of cells (of different experiments) will improve your statistical power to find new/different sub-populations? (Perhaps is specific of the methods, but I think they should already be present in each individual dataset)
Comments.UserDisplayName: llrs
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2018-02-19T22:59:48.890Z
Comments.Text: I am thinking of sampling cells from different part of the same organ as a proxy for spatial resolution, for instance. The ability of merging different datasets may give the opportunity to compare the different cells populations with added spatial heterogeneity distribution. But however, this is just an example.. in my case I was wondering if merging could add more information.
Comments.UserDisplayName: gc5
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-02T16:20:12.990Z
Comments.Text: How different is the experimental design? cellranger supports “reanalyse” across samples but I’m not sure this applies with different platforms generating the data.
Comments.UserDisplayName: Tom Kelly ケリー・トム
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-04T16:10:06.123Z
Comments.Text: @TomKelly they are generated with the same platform on 10X, but they are enriched differently using markers from FACS, so I think it is not possible.
Comments.UserDisplayName: gc5
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T20:46:54.923Z
Comments.Text: @TomKelly which is the tool within cellranger to merge different samples? I was looking at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/reanalyze but it seems to me it cannot be used to merge samples
Comments.UserDisplayName: gc5
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-07T12:15:19.970Z
Comments.Text: I remember attending a seminar where scientists merged data from different types of sc experiments (i think from the same platform though). Its long ago, so my memory might not be correct on this, but I do recall that it would be a functionality planned to be implemented in Seurat - although I haven't yet had any luck finding documentation on https://satijalab.org/seurat/. In addition I think that someone at the seminar mentioned using WGCNA to map batch effects. Since my memory is not clear on this subject I don't dare post this as an answer though!
Comments.UserDisplayName: Kasper Thystrup Karstensen
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-07T12:18:11.323Z
Comments.Text: This can possibly be of interest:
  https://www.ncbi.nlm.nih.gov/pubmed/29608177 - Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors
Comments.UserDisplayName: Kasper Thystrup Karstensen
Tags: <rna-seq><scrnaseq><batch-effects><merge>
Title: Is it possible to merge scRNAseq data from experiments with different design?
ThreadText: # Is it possible to merge scRNAseq data from experiments with different design?
  
  (Asked by: username_0 on 2018-02-19)
  
  I have 4 different single-cell RNAseq experiments, each one representing a different sample of cell type population. I'd like to merge them to a single dataset. However, different cell types are enriched in each sample, and different samples can represent also different conditions (control and disease).
  
  If I run a PCA on the merged dataset (without any processing of data) I find that the different sample's cells cluster separately. This could be due to biological and technical (i.e. batch effects) variation.
  
  Is it possible to merge these different samples while retaining a good amount of information or should they be analyzed separately?
  
  **Tags:** rna-seq, scrnaseq, batch-effects, merge
  
  > **Comments:**
  > **username_1**: Is there some samples (controls perhaps) in both experiments? What is your goal by merging them: compare a cell line of experiment A with a cell line in experiment B?  
  > **username_0**: No controls unfortunately. My goal by merging them is basically to find different sub-populations in the data and markers by exploiting a larger dataset. This data was presented to me after sequencing, and I think that it is only possible to use each experiment separately, but I'd like to be proven wrong - at least in some specific analysis.  
  > **username_1**: I'm not familiar enough with the methods for scRNA, sorry. But why do you think that increasing the number of cells (of different experiments) will improve your statistical power to find new/different sub-populations? (Perhaps is specific of the methods, but I think they should already be present in each individual dataset)  
  > **username_0**: I am thinking of sampling cells from different part of the same organ as a proxy for spatial resolution, for instance. The ability of merging different datasets may give the opportunity to compare the different cells populations with added spatial heterogeneity distribution. But however, this is just an example.. in my case I was wondering if merging could add more information.  
  > **username_2**: How different is the experimental design? cellranger supports “reanalyse” across samples but I’m not sure this applies with different platforms generating the data.  
  > **username_0**: @TomKelly they are generated with the same platform on 10X, but they are enriched differently using markers from FACS, so I think it is not possible.  
  > **username_0**: @TomKelly which is the tool within cellranger to merge different samples? I was looking at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/reanalyze but it seems to me it cannot be used to merge samples  
  > **username_3**: I remember attending a seminar where scientists merged data from different types of sc experiments (i think from the same platform though). Its long ago, so my memory might not be correct on this, but I do recall that it would be a functionality planned to be implemented in Seurat - although I haven't yet had any luck finding documentation on https://satijalab.org/seurat/. In addition I think that someone at the seminar mentioned using WGCNA to map batch effects. Since my memory is not clear on this subject I don't dare post this as an answer though!  
  > **username_3**: This can possibly be of interest: https://www.ncbi.nlm.nih.gov/pubmed/29608177 - Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors  
  
  ---
  
  ## 3 answers
  
  ### Answer by username_2 on 2018-06-06. Score: 2 | Accepted answer
  
  It’s right there on the [cellranger manual](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger):
  
  ```
  #aggregate results of counts for separate samples
  cellranger aggr
  #analyse the combined results
  cellranger reanalyze
  
  ```
  
  Note: I’m not sure this applies to the above case where batch effects could be an issue. However, this is how the analysis is performed for 10X data from multiple runs in principle.
  
  [cellranger aggr](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/aggregate)
  
  [cellranger reanalyze](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/reanalyze)
  
  > **Comments:**
  > **username_0**: Adding to this answer months later: it does not work with different designs, it just runs depth normalization but it can be severely affected by batch effects.  
  > **username_2**: Yes batch effects must be considered. Since this question was specifically about combining datasets, that is what was described here. This is the first step to adjust for batch effects, there are several methods to do this on an aggregate dataset and there are separate questions on this already (these assume some common cells between samples or it will overfit). I recommend not to use downsampling but aggregate raw. Depth normalisation does not remove batch effects or background noise in each sample.  
  > **username_0**: I agree. As you said, methods like ComBat are not suitable for experiments with different design because they require a balanced design (some common cells between samples). Thanks.  
  > **username_2**: The 3.0 release of the Seurat R package (currently in alpha) will have extensive support for combining data from different experiments.  
  
  ---
  
  ### Answer by username_4 on 2018-06-07. Score: 2
  
  See [this paper from the Marioni group](https://www.ncbi.nlm.nih.gov/pubmed/29608177), where they propose a method for correcting batch effects between single cell sequencing experiments when each experiment contains different sub-populations:
  
  > Large-scale single-cell RNA sequencing (scRNA-seq) data sets that are
  > produced in different laboratories and at different times contain
  > batch effects that may compromise the integration and interpretation
  > of the data. Existing scRNA-seq analysis methods incorrectly assume
  > that the composition of cell populations is either known or identical
  > across batches. We present a strategy for batch correction based on
  > the detection of mutual nearest neighbors (MNNs) in the
  > high-dimensional expression space. Our approach does not rely on
  > predefined or equal population compositions across batches; instead,
  > it requires only that a subset of the population be shared between
  > batches. We demonstrate the superiority of our approach compared with
  > existing methods by using both simulated and real scRNA-seq data sets.
  > Using multiple droplet-based scRNA-seq data sets, we demonstrate that
  > our MNN batch-effect-correction method can be scaled to large numbers
  > of cells
  
  
  ---
  
  ### Answer by username_5 on 2019-06-27. Score: 1
  
  Have you tried seurat3? Here is the reference:
  <https://satijalab.org/seurat/v3.0/pancreas_integration_label_transfer.html>
  
  
  ---
  
  