Batch effects in omics data are notoriously common technical variations unrelated to study objectives, and may result in misleading outcomes if uncorrected, or hinder biomedical discovery if over-corrected. Assessing and mitigating batch effects is crucial for ensuring the reliability and reproducibility of omics data and minimizing the impact of technical variations on biological interpretation. In this review, we highlight the profound negative impact of batch effects and the urgent need to address this challenging problem in large-scale omics studies. We summarize potential sources of batch effects, current progress in evaluating and correcting them, and consortium efforts aiming to tackle them.

Characterization and integration of the genome, epigenome, transcriptome, proteome and metabolome of different datasets is difficult owing to a lack of ground truth. Here we develop and characterize suites of publicly available multi-omics reference materials of matched DNA, RNA, protein and metabolites derived from immortalized cell lines from a family quartet of parents and monozygotic twin daughters. These references provide built-in truth defined by relationships among the family members and the information flow from DNA to RNA to protein. We demonstrate how using a ratio-based profiling approach that scales the absolute feature values of a study sample relative to those of a concurrently measured common reference sample produces reproducible and comparable data suitable for integration across batches, labs, platforms and omics types. Our study identifies reference-free ‘absolute’ feature quantification as the root cause of irreproducibility in multi-omics measurement and data integration and establishes the advantages of ratio-based multi-omics profiling with common reference materials.

Certified RNA reference materials are indispensable for assessing the reliability of RNA sequencing to detect intrinsically small biological differences in clinical settings, such as molecular subtyping of diseases. As part of the Quartet Project for quality control and data integration of multi-omics profiling, we established four RNA reference materials derived from immortalized B-lymphoblastoid cell lines from four members of a monozygotic twin family. Additionally, we constructed ratio-based transcriptome-wide reference datasets between two samples, providing cross-platform and cross-laboratory ‘ground truth’. Investigation of the intrinsically subtle biological differences among the Quartet samples enables sensitive assessment of cross-batch integration of transcriptomic measurements at the ratio level. The Quartet RNA reference materials, combined with the ratio-based reference datasets, can serve as unique resources for assessing and improving the quality of transcriptomic data in clinical and biological settings.

Background: RNA preparations contaminated with genomic DNA (gDNA) are frequently disregarded by RNA-seq studies. Such contamination may generate false results; however, their effect on the outcomes of RNA-seq analyses is unknown. To address this gap in our knowledge, here we added different concentrations of gDNA to total RNA preparations and subjected them to RNA-seq analysis. Results: We found that the contaminating gDNA altered the quantification of transcripts at relatively high concentrations. Differentially expressed genes (DEGs) resulting from gDNA contamination may therefore contribute to higher rates of false enrichment of pathways compared with analogous samples lacking numerous DEGs. A strategy was developed to correct gene expression levels in gDNA-contaminated RNA samples, which assessed the magnitude of contamination to improve the reliability of the results. Conclusions: Our study indicates that caution must be exercised when interpreting results associated with low-abundance transcripts. The data provided here will likely serve as a valuable resource to evaluate the influence of gDNA contamination on RNA-seq analysis, particularly related to the detection of putative novel gene elements.