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Ambient RNA analysis reveals misinterpreted and masked cell types in brain single-nuclei datasets

ABSTRACT: Ambient RNA contamination in single-cell RNA sequencing (RNA-seq) is a significant problem, but its consequences are poorly understood. Here, we show that ambient RNAs in brain single-nuclei RNA-seq can have a nuclear or extra-nuclear origin, and each origin results in a distinct gene set signature. Both ambient RNA signatures are predominantly neuronal and we find that some previously annotated neuronal cell types are distinguished by ambient RNA. Strikingly, we also detect pervasive neuronal ambient RNA contamination in all glial cell types unless glia and neurons are physically separated prior to sequencing. We demonstrate that this contamination can be removed in silico using existing tools. We also show that previous annotations of immature oligodendrocytes are likely glial cells contaminated with ambient RNAs. After ambient RNA removal, we can detect extremely rare committed oligodendrocyte progenitor cells, which were infrequently annotated in previously published adult human brain datasets. Together, these results provide an in-depth analysis of ambient RNA contamination in brain single-cell datasets.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE198951 | GEO | 2022/10/19

REPOSITORIES: GEO

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Project description:Autism spectrum disorder (ASD) is a common, highly heritable neurodevelopmental condition characterized by marked genetic heterogeneity. Thus, a fundamental question is whether autism represents an aetiologically heterogeneous disorder in which the myriad genetic or environmental risk factors perturb common underlying molecular pathways in the brain. Here, we demonstrate consistent differences in transcriptome organization between autistic and normal brain by gene co-expression network analysis. Remarkably, regional patterns of gene expression that typically distinguish frontal and temporal cortex are significantly attenuated in the ASD brain, suggesting abnormalities in cortical patterning. We further identify discrete modules of co-expressed genes associated with autism: a neuronal module enriched for known autism susceptibility genes, including the neuronal specific splicing factor A2BP1 (also known as FOX1), and a module enriched for immune genes and glial markers. Using high-throughput RNA sequencing we demonstrate dysregulated splicing of A2BP1-dependent alternative exons in the ASD brain. Moreover, using a published autism genome-wide association study (GWAS) data set, we show that the neuronal module is enriched for genetically associated variants, providing independent support for the causal involvement of these genes in autism. In contrast, the immune-glial module showed no enrichment for autism GWAS signals, indicating a non-genetic aetiology for this process. Collectively, our results provide strong evidence for convergent molecular abnormalities in ASD, and implicate transcriptional and splicing dysregulation as underlying mechanisms of neuronal dysfunction in this disorder. To identify potential A2BP1-dependent differential splicing events in ASD brain, we performed high-throughput RNA sequencing (RNA-Seq) on three autism samples with significant downregulation of A2BP1 (average fold change by quantitative RT-PCR = 5.9) and three control samples with average A2BP1 levels. The list of potential A2BP1-depending differential splicing events in ASD is given in the Supplementary file linked at the foot of this record.

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Ambient RNA analysis reveals misinterpreted and masked cell types in brain single-nuclei datasets

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