Batch correcting for different 10X 3’ assay chemistry versions
October 17, 2020
I’ve recently had the chance to work on some single-cell RNA-seq (scRNAseq) datasets generated with the 10X Genomics 3’ assay. The way I’ve been analyzing this data is by running the UMI counts through the `cellranger count` pipeline and loading the resulting filtered feature-barcode matrices as input for a Seurat object in R.
Seurat, by the way, is an incredibly well-documented and easy-to-use R package designed specifically for scRNAseq analysis. I was intimidated by the idea of analyzing single-cell data, but Seurat got me started in no time and before I knew it, I was identifying cluster markers and looking at how differentially expressed genes could help explain changes seen from diagnosis to relapse in Hodgkin’s Lymphoma cases. So cool!
One of the unique aspects of Seurat is its approach to batch-correction (or “integration” in Seurat’s terms) which uses a combination of Canonical Correlation Analysis (conceptually similar to the Needleman-Wunsch algorithm) and mutual nearest neighhours (MNN). The gist of their approach aims to identify shared cell states that are present across different datasets -- these “anchors” allow for the transfer information from one dataset to another, resulting in a single harmonized reference.
Before using Seurat, I wasn’t familiar with any other batch-correction methods for scRNAseq, but a colleague pointed out that a MNN-based approach by Haghverdi et al. is fairly commonly used in the field. Since I was still new to the topic and wanted to make sure I was using the best method for my analysis, I did some research to see if there were any studies comparing the two methods, and to see perhaps if even other methods might be more suitable.
A very comprehensive study by Tran et al. was published in Genome Biology earlier this year, and examines the performance of 14 scRNAseq batch-correction methods applied to 10 datasets. The study looks at diverse scenarios, including combining batches with: identical cell types but different technologies, non-identical cell types, more than two batches, “big data” and simulated data. No one tool was the winner, but Seurat3, Harmony and LIGER performed consistently well across the majority of datasets.
So I continued using Seurat for all my analyses and felt good about it. Eventually, I started to examine individual genes exhibiting significant changes in expression between groups of interest (i.e. diagnosis vs. relapse cases), when I came across JUND, a proto-oncogene that appeared to be highly expressed at relapse but very lowly expressed at diagnosis. Surprisingly, the expression pattern was similar across all clusters and thus could not be linked to a given cell type. This didn’t make sense biologically since such extreme changes in gene expression would need to come from a specific immunophenotype and not all the cells.
I then looked at expression of JUND at the sample level, and noticed that in addition to all of the diagnostic samples, a handful of the relapse samples were not expressing JUND. When I presented these results to my lab, the technician who runs the sequencing experiments, recalled these samples were prepared using an older version of the 3’ assay (V2). Indeed, the 10X website shows data indicating that their 3’ V3 assay results in increased UMI counts and numbers of genes per cell compared to their 3’ V2 assay, which I confirmed with my own data.
So what’s the best way to correct for differences in chemistry, and how can one account for gene dropouts?
Well, I already knew the first part of the answer (batch-correction), but I wasn’t sure how to address the gene drop out problem, which unaccounted for, would inevitably skew my differential expression analyses. I went on a call with my team to discuss this issue with 10X, and discovered they have exactly a solution for us: Aggregating Libraries With Different Chemistry Versions... Or did they?
The `cellranger aggr` pipeline, by default, aggregates outputs from multiple runs of the cellranger count pipeline by sub-sampling reads from higher-depth cells until all cells on average have an equal numbers of cells. This is essentially 10X’s way of normalizing the data for sequencing depth, but is not ideal since a potentially large amount of data is discarded right off the bat. The alternative normalization approach (e.g. in Seurat) fits a model (e.g. regularized negative binomial regression) to the UMI counts. Thus, a transformation is applied to the data instead of physically removing the reads.
In addition to normalization, the `aggr` pipeline can carry out an additional but optional step that corrects for different chemistry versions given batch information. Here, batches are referring to the libraries sequenced with the same assay version. While I was already a little skeptical of using this approach since the normalization step throws away potentially valuable data, the 10X representative made me even more doubtful by showing me “before” and “after” UMAP visualizations that were less-than-convincing. She also noted that the batch-correction step is only designed for combining V2 and V3 chemistries, and not intended to discern batch effects between different runs. Noneless, I kept an open mind.
After our call, I read through all the documentation on this topic, and was surprised to learn that the batch-correction method specifically designed for combining V2 and V3 chemistries was the MNN approach. As you may recall, this was the same method I mentioned earlier that is commonly used for batch-correction in scRNAseq analysis. Wait a second… so correcting for different chemistries just means I can correct for different batches? This actually makes a lot of sense, since a batch needs to be prepared with the same chemistry, so in reality I was already indirectly correcting for the chemistries by correcting for technical batches.
There are a few takeaway messages here:
- To correct for differences in chemistry between batches, you can use your favourite batch-correction method. You don’t and probably shouldn’t have to use 10X’s aggr pipeline since you’ll lose lots of reads, and the correction step won’t be that good (MNN doesn’t perform that well compared to a number of other scRNAseq batch correction methods).
- Once you’ve corrected for different chemistry versions (and batches), you’re still going to see gene dropouts. This is actually a major unsolved problem in scRNAseq analyses, but there are a few approaches that might help:
- Do your homework! Companies love promoting their products, even if it may not be the right solution for (but they will make it sound like it is). By doing my research, I saved myself from re-doing all my analyses with a worse pipeline and would probably end up being even more confused than when I started.