BackgroundVariation in cell composition can dramatically impact analyses in bulk tissue samples. A commonly employed approach to mitigate this issue is to adjust statistical models using estimates of cell abundance derived directly from omics data. While an arsenal of estimation methods exists, the applicability of these methods to brain tissue data and whether or not cell estimates can sufficiently account for confounding cellular composition has not been adequately assessed.MethodsWe assessed the correspondence between different estimation methods based on transcriptomic (RNA sequencing, RNA-seq) and epigenomic (DNA methylation and histone acetylation) data from brain tissue samples of 49 individuals. We further evaluated the impact of different estimation approaches on the analysis of H3K27 acetylation chromatin immunoprecipitation sequencing (ChIP-seq) data from entorhinal cortex of individuals with Alzheimer’s disease and controls.ResultsWe show that even closely adjacent tissue samples from the same Brodmann area vary greatly in their cell composition. Comparison across different estimation methods indicates that while different estimation methods applied to the same data produce highly similar outcomes, there is a surprisingly low concordance between estimates based on different omics data modalities. Alarmingly, we show that cell type estimates may not always sufficiently account for confounding variation in cell composition.ConclusionsOur work indicates that cell composition estimation or direct quantification in one tissue sample should not be used as a proxy to the cellular composition of another tissue sample from the same brain region of an individual—even if the samples are directly adjacent. The highly similar outcomes observed among vastly different estimation methods, highlight the need for brain benchmark datasets and better validation approaches. Finally, unless validated through complementary experiments, the interpretation of analyses outcomes based on data confounded by cell composition should be done with great caution, and ideally avoided all together.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13073-023-01195-2.