Navigating RNA-seq considerations and challenges

Navigating RNA sequencing (RNA-seq): considerations and challenges

RNA sequencing (RNA-seq) has become immensely popular and indispensable in modern biological research due to its versatility and depth of information. By providing a comprehensive snapshot of the transcriptome, RNA-seq enables the quantification of gene expression levels, detection of alternative splicing events, and identification of non-coding RNAs. Its ability to capture dynamic changes in gene expression under different conditions or treatments makes RNA-seq invaluable for studying various biological processes, including development, disease mechanisms, and drug responses. Furthermore, advancements in sequencing technology and data analysis tools have made RNA-seq more accessible and cost-effective, further fuelling its widespread adoption across diverse fields of biology. As researchers navigate RNA sequencing, they must consider the advantages and limitations of the range of RNA-seq approaches, including coverage, sensitivity, tissue type, and experimental context. Understanding RNA-seq analysis nuances is crucial for maximising sequencing data utility and unravelling gene expression regulation complexities. 

Balancing sensitivity and noise in RNA-seq analysis 

RNA-seq, while powerful, faces challenges in sensitivity and noise. The balance between sensitivity and noise is critical in RNA-seq analysis. Technical limitations in library preparation and high sequencing depth requirements can lead to difficulties in detecting low-abundance transcripts, potentially underestimating or omitting important biological signals. Even when sequencing at a depth high enough to capture low frequency transcripts, the associated noise buildup can mask the transcripts that are of most importance. High background noise from sequencing errors, PCR amplification biases, and other technical artefacts can obscure genuine transcriptomic differences, making it challenging to distinguish true biological variability from experimental noise

The dilemma of choosing the right RNA-seq methodology: potential pitfalls and challenges 

Selecting the appropriate RNA-seq technique is critical, as each method comes with its own set of advantages and disadvantages. Opting for traditional RNA-seq methods, the average gene expression levels across the entire sample are assessed at the moment of cDNA conversion. This may obscure subtle differences in gene expression overtime and between states. To improve resolution, nascent RNA-seq specifically profiles newly transcribed RNA molecules across a given time window, which provides insights into functional transcriptome dynamics, but requires specialised experimental protocols and may have lower coverage compared to traditional methods. Moreover, the choice between standard and nascent RNA-seq depends on the research question and experimental objectives. Failure to consider these factors can lead to misinterpretation of results and limited insights into gene regulation dynamics. 

Challenges in tissue selection for RNA and epigenetic analysis: 

Selecting the appropriate tissue type poses a pivotal challenge in RNA and epigenetic data analysis, profoundly influencing the interpretation and applicability of findings. Tissue degradation is a major concern for RNA-seq experiments. High quality tissue is required due to the instability of RNA, which can degrade rapidly over time in low quality samples. Certain tissue types are therefore unsuitable for RNA-seq analysis. Brain tissue is a prominent example of this, as it must be collected quickly post-mortem to avoid any degradation, which often cannot happen. Formalin-Fixed Paraffin Embedded (FFPE) samples also display this issue, where the fixation process causes RNA fragmentation and modifications leading to biased transcriptome profiles and inaccurate gene expression quantification. Artefacts like cross-linking may interfere with sequencing processes. Variability in sample quality and quantity can introduce batch effects and confounding factors, complicating data interpretation. 

Tissue-specific variations in gene expression and epigenetic regulation intricately define cellular identity and function. Combining transcriptomic and epigenetic data can bring to light the complex regulatory mechanisms that control tissue specificity. For example, insights in neurological disorders can be unveiled by scrutinising gene expression and epigenetic patterns in brain tissue, and looking at the same data from adipose tissue sheds light on metabolic diseases.  

Integrating RNA and epigenetic data, although promising, introduces complexities when combining disparate data sets where areas with reduced coverage in either data set are not represented and therefore cannot contribute to understanding biological significance. Nonetheless, merging these data types offers a comprehensive framework for exploring how epigenetic modifications influence gene expression dynamics

Modern studies and techniques are increasingly interested in multiomics, the study of multiple informational modalities. RNA-seq, amongst other techniques, can be combined with separate modalities to give enhanced information and generate new insight about how different biological systems interact. Once these separate data sets have been generated, they must be integrated. Integrating multiomics data presents a formidable challenge due to the inherent complexity of biological systems. Combining diverse molecular layers such as genomics, transcriptomics, epigenomics, proteomics, and metabolomics requires sophisticated computational methods and analytical frameworks to unravel the intricate interactions and regulatory networks at play. The integration process involves addressing data heterogeneity, normalisation, and integration across omics platforms, each with its own set of technical challenges and biases. Moreover, deciphering the causal relationships and biological significance of multiomics findings requires careful interpretation and validation using complementary experimental approaches. Despite these challenges, multiomics integration holds immense promise for advancing our understanding of complex biological phenomena and disease processes, offering insights into molecular mechanisms and potential therapeutic targets. 

Use duet multiomics solution evoC to simultaneously explore epigenetic and transcriptomic regulation 

Integration of disparate data sets represents a computational burden on any multiomics experiment that uses several techniques in parallel, such as the common combination of RNA-seq and assay for transposase-accessible chromatin with sequencing (ATAC-seq). However, this burden can be overcome by technologies that generate multiple layers of information simultaneously from the same piece of genetic material. duet multiomics solution evoC generates both genetic and epigenetic information simultaneously on the same DNA fragment, removing the need for integrating those data layers. It captures the full genetic sequence of a sample, as well as resolving the DNA methylation marks 5‑methylcytosine (5mC) and 5‑hydroxymethylcytosine (5hmC).  

While RNA-seq offers valuable insights into gene expression dynamics, whole-genome sequencing (WGS) combined with DNA methylation analysis provides complementary information that enhances our understanding of genomic regulation and epigenetic modifications. Dr. Emily Hodges, a renowned expert in genomics and epigenetics at the Department of Molecular Biology, Massachusetts Institute of Technology (MIT), emphasises the advantages of WGS and DNA methylation analysis, stating, By integrating whole-genome sequencing with DNA methylation analysis, we gain a comprehensive view of genetic and epigenetic landscapes, revealing the intricate interplay between genomic regulation and epigenetic modifications, thus deepening our understanding of cellular processes and disease mechanisms 

Moreover, the discrete identification of 5mC and 5hmC reveals insights into how epigenetic modifications regulate gene expression, providing valuable context for interpreting genomic data. As these two epigenetic marks inhibit or promote gene expression respectively, identifying both allows researchers to build a model that accurately predicts gene expression, without having to deal with the complications and intricacies of isolating and measuring RNA transcripts. This is achieved on top of all the other benefits that come from genetic and methylation sequencing, in a truly multiomic solution. By training predictive models on integrated multiomics datasets, researchers can identify key regulatory elements and epigenetic signatures associated with gene expression patterns, facilitating the discovery of novel regulatory mechanisms and biomarkers for disease prognosis and therapeutic targeting. 

Overall, the integration of genetic information with discrete mC and hmC data for gene expression analysis using duet evoC offers several advantages: 

  1. Comprehensive Insights into gene regulation: integrated analysis of WGS, mC, and hmC data provides a comprehensive view of gene regulation mechanisms, enabling researchers to uncover the complex interplay between genetic and epigenetic factors in shaping gene expression profiles. 
  2. Identification of regulatory elements: by correlating genetic variations with DNA methylation and hydroxymethylation patterns, researchers can identify cis-regulatory elements and trans-acting factors that modulate gene expression, shedding light on the regulatory landscape of the genome.
  3. Predictive modelling of gene expression: computational tools supporting integrated analysis of WGS, mC, and hmC data facilitate the development of predictive models for gene expression based on genetic and epigenetic features. These models enable researchers to identify key regulatory elements and epigenetic signatures associated with gene expression patterns, offering insights into disease mechanisms and potential therapeutic targets. 

In summary, duet multiomics solution evoC offers a powerful approach for unravelling gene expression regulation and understanding the molecular mechanisms underlying complex traits and diseases. By leveraging multiomics data integration and predictive modelling techniques, researchers can accelerate discoveries in genomics and epigenomics research and pave the way for personalised medicine applications. 


  1. Shishkin AA, Giannoukos G, Kucukural A, et al. Simultaneous generation of many RNA-Seq libraries in a single reaction. Nat Methods. 2015;12(4):323-325. doi:10.1038/nmeth.3313 
  2. Kivioja T, Vähärautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, Taipale J. Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2012 Jul;9(1):72-4. doi: 10.1038/nmeth.1778. Epub 2011 Nov 13. PMID: 22081019. 
  3. La Manno G, Soldatov R, Zeisel A, et al. RNA velocity of single cells. Nature. 2018;560(7719):494-498. doi:10.1038/s41586-018-0414-6 
  4. Boudoures AL, Saben J, Drury A, et al. Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. Dev Biol. 2017;426(1):126-138. doi:10.1016/j.ydbio.2017.04.023 
  5. Milani P, Escalante-Chong R, Shelley BC, et al. Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells. Sci Rep. 2016;6:25474. Published 2016 May 6. doi:10.1038/srep25474 
Cambridge Epigenetix is now biomodal