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Total RNA Sequencing Breakthroughs Transform Research Accessibility

RNA Research for All: How Advances in Total RNA Sequencing are Transforming Accessibility

The study of the transcriptome has revolutionized our understanding of gene expression and regulation across biological systems. RNA sequencing (RNA-Seq) has emerged as a fundamental approach to this analysis, providing unprecedented insights into cellular function in both health and disease contexts. Despite its importance, comprehensive transcriptome analysis has historically been limited by technical challenges, high costs, and complex workflows that placed it beyond the reach of many research laboratories.1 

Recent technological advancements in Total RNA Sequencing are making comprehensive transcriptome analysis more accessible, enabling researchers across diverse fields to leverage this powerful approach. Unlike traditional messenger RNA (mRNA) sequencing that primarily captures polyadenylated transcripts, Total RNA Sequencing offers a broader view of the transcriptome, including both coding and non-coding RNA species that play critical roles in cellular regulation.2 ​Broad Clinical Labs has been at the forefront of this transformation, pioneering innovative approaches that combine globin and ribosomal RNA (rRNA) depletion with unique molecular identifiers (UMIs) to dramatically enhance transcript detection capabilities while overcoming long-standing technical barriers. ​These advances represent more than incremental improvements – they signal a paradigm shift in how we approach transcriptome research, who can participate in this research, and what questions can be answered.3 

The Evolution of RNA Sequencing Technologies 

RNA sequencing has undergone tremendous evolution since its inception. Early approaches focused almost exclusively on mRNA with polyadenylated (poly-A) tails, capturing only a fraction of the transcriptome and missing critical regulatory RNA species. These methods imposed strict requirements on sample quality and quantity, showed bias toward abundant transcripts, and involved complex workflows requiring specialized expertise. Meanwhile, the prohibitive costs made large-scale studies inaccessible to many research groups. 

Today’s RNA sequencing technologies represent a transformative advancement. Modern approaches have overcome many of these limitations through innovations in library preparation chemistry, molecular biology techniques, and sequencing platforms. Combined ribosomal RNA (rRNA) and globin depletion methods have dramatically improved the signal-to-noise ratio in samples and increased the sensitivity for low abundance transcripts. Moreover, unique molecular identifiers (UMIs) enable more accurate quantification of transcript abundance. Next-generation sequencing platforms have simultaneously increased throughput while decreasing per-base costs, making comprehensive transcriptome analysis more economically feasible for research groups. 

Gaining a Broader View of Biology with Total RNA Sequencing 

The most significant advantage of Total RNA Sequencing is its comprehensive coverage of the transcriptome. Rather than focusing exclusively on mRNA, this approach captures the full diversity of RNA species, including microRNA, circular RNAs, long-non-coding RNA, and enhancer RNAs, providing a complete picture of gene expression dynamics regardless of polyadenylation status.4 This broader view enables researchers to explore regulatory networks and functions that would remain elusive with traditional approaches. The detection of alternative splicing events, which play crucial roles in cellular differentiation and disease processes, is particularly enhanced with Total RNA Sequencing approaches.5 

Modern Total RNA Sequencing protocols have dramatically relaxed sample requirements, making the technology accessible for challenging research contexts. Key improvements include lower input requirements, success with partially degraded samples, and compatibility with challenging sample types including blood and fresh frozen tissue. These advancements have significantly reduced limitations associated with sample availability and quality, thereby expanding the potential applications of Total RNA Sequencing to new research areas.  

Expanding Access to Total RNA Sequencing  

A key factor in expanding access to Total RNA Sequencing has been the development of automated workflows. Fully automated platforms maximize reproducibility, reducing technical artifacts and batch effects that previously complicated data interpretation. The scalability improvements made possible through automation have transformed Total RNA Sequencing from a boutique technology applicable to dozens of samples, into a high-throughput approach capable of processing thousands of samples with consistent quality and performance.​ ​Total RNA Sequencing has been the development of automated workflows. Fully automated platforms maximize reproducibility, reducing technical artifacts and batch effects that previously complicated data interpretation. The scalability improvements made possible through automation have transformed Total RNA Sequencing from a boutique technology applicable to dozens of samples, into a high-throughput approach capable of processing thousands of samples with consistent quality and performance. 

Perhaps the most important factor in democratizing access to Total RNA Sequencing has been the dramatic reduction in costs. Higher data yield per sequencing run with platforms like NovaSeq X has significantly lowered per-sample sequencing costs. Additionally, optimized library preparation workflows with fewer steps and reagents have reduced both labor and material expenses, while multiplexing capabilities maximize instrument utilization. This substantial decrease in cost per sample has enabled more research groups to include RNA-Seq as part of their experimental toolkit. 

“The democratization of transcriptomics through these technological advancements is accelerating the pace of discovery across medical research, enabling insights that were previously inaccessible to many research teams,” notes Niall Lennon, CSO and Chair at Broad Clinical Labs. “We’re seeing a transformation in who can participate in cutting-edge genomic research.” 

Diverse Research Applications 

The enhanced capabilities of Total RNA Sequencing have made it an invaluable tool for biomarker discovery and precision medicine applications. By capturing the full spectrum of RNA species, researchers can identify novel transcript biomarkers that might be missed with traditional approaches. RNA signatures derived from Total RNA-Seq have proven particularly valuable for cancer classification and treatment response prediction, neurodegenerative disease characterization, autoimmune disorder profiling, and cardiovascular risk assessment. With these advanced capabilities, RNA signatures are now being actively implemented as diagnostic tools in clinical practice, enabling more accurate disease detection and monitoring across healthcare settings.6 

Beyond its clinical applications, Total RNA Sequencing has transformed our understanding of gene regulation by illuminating the complex networks of non-coding RNAs that influence cellular processes. These non-coding molecules, historically dismissed, are now recognized as critical components of the cellular machinery. Total RNA Sequencing has proven especially valuable for mapping interactions between coding and non-coding RNA species, identifying regulatory elements controlling gene expression patterns, and characterizing alternative splicing patterns in development and disease.6 

Notably, transcriptional signatures often provide deeper insight into disease mechanisms than DNA sequencing alone, reflecting the functional activity of cells rather than just their genetic potential. As researchers continue to analyze these comprehensive RNA profiles, the resulting insights are fundamentally reshaping our understanding of cellular biology and opening new avenues for therapeutic intervention, particularly through the discovery of potential approaches targeting regulatory RNAs rather than traditional protein targets. 

Proven Advantages Through Comparative Analysis 

To illustrate the practical impact of advances in Total RNA Sequencing, we highlight a case study that compares with traditional mRNA sequencing approaches. In a comprehensive evaluation conducted by Broad Clinical Labs, blood samples processed with their Total RNA workflow (including globin and rRNA depletion plus UMI incorporation) consistently demonstrated ​​​​superior transcript detection capabilities compared to standard mRNA sequencing methods. This enhanced sensitivity can be leveraged to identify low-abundance transcripts, revealing previously undetectable elements of gene expression and enabling researchers to draw more accurate conclusions about cellular activity and function. 

Broad Clinical Lab’s modern Total RNA Sequencing approach has overcome sample quality limitations through innovative chemistry and workflow improvements, enabling the successful sequencing of lower quality RNA inputs (RIN > 3.5) while also effectively processing limited sample quantities (≥ 500ng total). Furthermore, specialized protocols developed for challenging sample types have dramatically expanded the range of research contexts where RNA-Seq can be successfully applied. 

The Road Ahead for Transcriptomics 

Looking ahead, the evolution of transcriptomics will be defined by its integration with diverse ‘omics approaches. As researchers increasingly adopt multi-omics strategies, Total RNA Sequencing data gains exponential value when analyzed alongside genomic variants, epigenetic modifications, protein expression patterns, and metabolic profiles. This convergence embodies the essence of systems biology, which captures the dynamic interplay between different molecular layers. By leveraging multi-omics datasets, scientists can uncover regulatory mechanisms that would remain elusive through isolated analyses, advancing our understanding of biological systems and accelerating the development of clinical applications. 

Conclusion 

The most significant impact of recent advances in Total RNA Sequencing technology is the increased attainability of whole transcriptome analysis. By dramatically reducing technical barriers, cost constraints, and sample limitations, powerful RNA analysis tools are now more accessible to researchers across diverse fields and institutions. This wider access has fundamentally transformed how we study gene expression and regulation, expanding the universe of possible research questions, sample types, and experimental designs that can incorporate Total RNA Sequencing approaches. 

For researchers ready to leverage these advancements in their own work, comprehensive Total RNA Sequencing services are available through industry-leading providers like Broad Clinical Labs, offering standardized, high-quality transcriptome analysis with expert support and interpretation. Visit https://broadclinicallabs.org/rna-sequencing/ to learn more.  

 

References: 

  1. Wang, Z et al. “RNA-Seq: a revolutionary tool for transcriptomics.” Nature Reviews Genetics. (2023) 24(7). doi:10.1038/s41576-023-00571-5 
  2. Potemkin, N. et al. “A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq.” Sci Rep. (2022) 12(12):621. doi: 10.1038/s41598-021-04209-4 
  3. Conesa, A. et al. “A survey of best practices for RNA-seq data analysis.” Genome Biology. (2022) 23(1). doi:10.1186/s13059-022-02601-5 
  4. Casamassimi, A. et al. “Transcriptome Profiling in Human Diseases: New Advances and Perspectives.” International Journal of Molecular Sciences. (2017) 18(1652).  doi:10.3390/ijms18081652 
  5. Stark, R. et al. “RNA sequencing: the teenage years.” Nature Reviews Genetics. (2021) 22(7). doi:10.1038/s41576-021-00370-8 
  6. Byron, S et al. “Translating RNA sequencing into clinical diagnostics: opportunities and challenges” Nat Rev Genet. (2016) 17(5):257-271. doi: 10.1038/nrg.2016.1 

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Sean Hofherr

Chief of Clinical Strategy and Product Development, Broad Clinical Labs

Sean Hofherr is dual board certified by ABMGG in Clinical Biochemical Genetics and Clinical Molecular Genetics. Sean serves as the Chief of Clinical Strategy and Product Development at Broad Clinical Labs. In this role at BCL, Sean is able to leverage his extensive experience to guide the clinical vision and delivery across the organization. Sean most recently served as the Chief Operating Office at Fabric Genomics, which focuses on the use of AI and Bioinformatics for Clinical Interpretation of whole genome sequencing. Prior to Fabric, Sean was the Chief Scientific Officer and CLIA Director at the commercial reference laboratory, GeneDx.

Sean received his B.S. degree in Microbiology and Cell Sciences from the University of Florida before earning his Ph.D. in Molecular and Human Genetics from Baylor College of Medicine. Sean completed clinical fellowships in Clinical Biochemical Genetics and Clinical Molecular Genetics at the Mayo Clinic.

Danielle Perrin

Chief of Staff, Broad Clinical Labs

As Broad Clinical Labs’ Chief of Staff, Danielle Perrin advises and supports colleagues on the executive leadership team in BCL’s strategic planning and execution. She builds and leads new organizational functions and processes and leads critical projects, as well as driving effective information flow, decision making, and execution throughout the organization. An operations leader with a business, engineering, and biology background and 20+ years of experience in the genomics field, Perrin has a track record of driving operational excellence and building and scaling both physical and business processes. During her career at Broad, which started in 2003 at the tail end of the Human Genome Project, Perrin has led laboratory operations and R&D teams in Broad’s Genomics Platform, as well as fulfilling senior advisory and leadership roles in the Broad Institute’s COO and CFO offices.

Perrin received her B.S. in Biology and M.E. in Biotechnology Engineering from Tufts University and her M.B.A. from the MIT Sloan School of Management.

Tim De Smet

Chief Commercial Officer, Broad Clinical Labs

As Chief Commercial Officer of Broad Clinical Labs, Tim De Smet leads BCL’s business development, alliance management, external project management, and customer support teams. A Broad Institute employee since 2008, De Smet has held leadership roles and managed teams of various sizes in Broad’s Genomics Platform and clinical lab, spanning laboratory operations, finance, and informatics, and has expertise in work design, financial modeling, and high scale laboratory and business operations.

De Smet received his B.S. in Biochemistry and M.B.A. from Northeastern University.

Jim Meldrim

Chief Technology Officer, Broad Clinical Labs

As Chief Technology Officer, Jim Meldrim sets the vision for Broad Clinical Labs’ informatics systems, including the hardware and software used for sample intake and tracking, data production, analysis, and delivery. Having held a variety of laboratory and informatics-focused leadership roles at Broad, spanning R&D and production operations, Meldrim has been a leader and innovator in the generation, management, and analysis of genomic data since 1999, beginning with sequencing data generation for the Human Genome Project.

Meldrim received his B.S. in Biology from Cornell University.

Sheila Dodge

Chief Operating Officer, Broad Clinical Labs

As Chief Operating Officer, Sheila Dodge leads Broad Clinical Labs’ process development and implementation activities, as well as lab operations, financial planning and operations, quality & compliance, and core business processes. A Six Sigma Black Belt with extensive experience in process development and high throughput genomics operations, Dodge is an expert in work design and in collaborating with a range of collaborators, scientists, engineers, and technology partners to rapidly integrate new technologies and operationalize innovations. A member of the Broad Institute since 2001, Dodge is an Institute Scientist and lectures at the MIT Sloan School of Management on operations, dynamic work design, and visual management techniques.

Dodge received her B.A. in biochemistry and molecular biology from Boston University and her master’s degree in biology from Harvard University. She earned her M.B.A. from MIT Sloan School of Management.

Heidi Rehm, Ph.D., FACMG

Chief Medical Officer and Clinical Laboratory Director, Broad Clinical Labs

Heidi Rehm is board-certified by ABMGG in Clinical Molecular Genetics and Genomics and serves as BCL’s Chief Medical Officer and Clinical Laboratory Director. She oversees BCL’s regulatory requirements, leads the clinical team performing genomic interpretation and variant analysis, and guides BCL’s efforts in genomic testing for clinical and research use. She is also an Institute Member of the Broad and co-director of the Medical and Population Genetics Program. Rehm is also the Chief Genomics Officer in the Department of Medicine and Genomic Medicine Unit Director at the Center for Genomic Medicine at Massachusetts General Hospital, working to integrate genomics into medical practice. She is a principal investigator of ClinGen, providing free and publicly accessible resources to support the interpretation of genes and variants. She co-leads both the Broad Center for Mendelian Genomics, focused on discovering novel rare disease genes, and the Matchmaker Exchange, which aids in gene discovery. She is Chair of the Global Alliance for Genomics and Health, a principal investigator of the Broad-LMM-Color All of Us Genome Center, co-leader of the Genome Aggregation Database (gnomAD), and a Board Member and Vice President of Laboratory Genetics for the American College of Medical Genetics and Genomics.

Rehm received her B.A. degree in molecular biology and biochemistry from Middlebury College before earning her M.S. in biomedical science from Harvard Medical School and Ph.D. in genetics from Harvard University. She completed her post-doctoral training with David Corey in neurobiology and a fellowship in clinical molecular genetics at Harvard Medical School.

Niall Lennon, Ph.D.

Chair and Chief Scientific Officer, Broad Clinical Labs

As Chair and Chief Scientific Officer of Broad Clinical Labs, Niall Lennon leads the team and sets the scientific and clinical vision for the organization. Dr. Lennon joined the Broad Institute in 2006 and has since contributed to the development of applications for every major massively parallel sequencing platform across a range of fields. In 2013 Dr. Lennon led the effort to establish a CLIA licensed, CAP-accredited clinical laboratory at the Broad Institute to facilitate return of results to patients and to support clinical trials. More recently, he has led efforts to achieve FDA approval for large-scale genomics projects (NIH’s All of Us Research Program) and for Broad’s own clinical diagnostic for COVID-19 testing operation, which returned 37+ million results to patients. Dr. Lennon is a principal investigator of the eMerge and All of Us projects, an Institute Scientist at Broad, Associate Director of Broad’s Gerstner Center for Cancer Diagnostics, and an adjunct professor of biomedical engineering at Tufts University, where he teaches Molecular Biotechnology.

Dr. Lennon received a Ph.D. in pharmacology from University College Dublin and completed his postdoctoral studies at Harvard Medical School and Massachusetts General Hospital. He holds an executive certificate in management from the MIT Sloan School of Management.