Question: Can viral gene expression be detected with the Single Cell Assays?
Answer: Detecting viral transcripts is possible with 10x Single Cell Gene Expression assays. Please see below for additional guidance on how this can be accomplished during both the wet-lab workflow and data analysis steps.
Our Single Cell GEX technology (both 3' and 5' assays) relies on poly-A tails to capture transcripts. The capture of the viral transcripts using our normal, unmodified workflow would require the transcripts to be poly-adenylated, either through their normal biological processes or by adding a poly-A tail to the viral vector.
10x customers have successfully used 10x Single Cell applications for detecting viral transcripts in virus-infected eukaryotic cells (please note that this viral RNA must be poly-adenylated for capture in either the 3' or 5' assay). There are no additional workflow modifications necessary to the protocol since "polyA mRNA." The detection of viral transcripts will depend on the abundance and adenylation states.
If the viral transcripts are not polyadenylated, or there is no PolyA tail on the vector transcripts, theoretically, the 5' protocol RT-primer can be modified using spiking-in a gene-specific primer sequence during the RT step or using random hexamers, but this is untested and unsupported. It might be easier to add a polyA signal to the expression vector. Please note that RT priming using a random hexamer would also produce cDNA from rRNA transcripts.
Note: We have not specifically tested these approaches; they are meant to be general guidelines. A pilot study is recommended before starting a large-scale experiment.
The following papers have successfully detected viral transcripts in 10x data and may be good resources for further study:
- Host-Viral Infection Maps Reveal Signatures of Severe COVID-19 Patients
- Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19
- Extreme heterogeneity of influenza virus infection in single cells
- Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity
- Analysis of cell-associated DENV RNA by oligo(dT) primed 5' capture scRNAseq
For Cell Ranger analysis, you will need to create a single custom reference with the viral sequence added to human reference FASTA and GTF.
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references For example, for an infected human cell line, if you run Cell Ranger with a combined transcriptome reference, the same barcode will show up in both matrices. It indicates that the GEM in question contained a human cell infected with the virus. Thus the human transcriptome was 'contaminated' with viral transcripts mapped back to the "EBV" reference.
Products: Single Cell Gene Expression, Single Cell Immune Profiling