Latest Developments in Rna Sequencing

🧬📘 𝗡𝗮𝗻𝗼𝗽𝗼𝗿𝗲 𝘀𝗲𝗾𝘂𝗲𝗻𝗰𝗶𝗻𝗴 𝗮𝘀 𝗮 𝗺𝘂𝗹𝘁𝗶-𝗮𝘁𝘁𝗿𝗶𝗯𝘂𝘁𝗲 𝘁𝗼𝗼𝗹 𝗳𝗼𝗿 𝗥𝗡𝗔 𝗾𝘂𝗮𝗹𝗶𝘁𝘆 📘🧬 As RNA therapeutics continue to expand across vaccines, cell therapy, and gene editing, analytical strategies must keep pace with increasingly complex quality requirements. In a recent publication, researchers from Genentech showed how nanopore direct RNA sequencing can support a more integrated assessment of critical quality attributes for both mRNA and sgRNA, within a single analytical workflow. 🔎 𝗧𝗵𝗲 𝘀𝘁𝘂𝗱𝘆 𝗵𝗶𝗴𝗵𝗹𝗶𝗴𝗵𝘁𝘀 𝘀𝗲𝘃𝗲𝗿𝗮𝗹 𝗶𝗺𝗽𝗼𝗿𝘁𝗮𝗻𝘁 𝗰𝗮𝗽𝗮𝗯𝗶𝗹𝗶𝘁𝗶𝗲𝘀: • 𝙁𝙪𝙡𝙡-𝙡𝙚𝙣𝙜𝙩𝙝 𝙨𝙚𝙦𝙪𝙚𝙣𝙘𝙚 𝙘𝙤𝙣𝙛𝙞𝙧𝙢𝙖𝙩𝙞𝙤𝙣 of mRNA without reverse transcription or PCR • 𝘿𝙞𝙧𝙚𝙘𝙩 𝙚𝙫𝙖𝙡𝙪𝙖𝙩𝙞𝙤𝙣 𝙤𝙛 𝙩𝙧𝙖𝙣𝙨𝙘𝙧𝙞𝙥𝙩 𝙞𝙣𝙩𝙚𝙜𝙧𝙞𝙩𝙮, including degradation-related fragmentation • 𝙋𝙤𝙡𝙮(𝘼) 𝙩𝙖𝙞𝙡 𝙘𝙝𝙖𝙧𝙖𝙘𝙩𝙚𝙧𝙞𝙯𝙖𝙩𝙞𝙤𝙣, with visibility into tail length distributions and heterogeneity • 𝟱′ 𝙘𝙖𝙥𝙥𝙞𝙣𝙜 𝙖𝙨𝙨𝙚𝙨𝙨𝙢𝙚𝙣𝙩 through a sequencing-based ligation strategy applied directly to native RNA molecules • 𝙁𝙪𝙡𝙡-𝙡𝙚𝙣𝙜𝙩𝙝 𝙨𝙜𝙍𝙉𝘼 𝙨𝙚𝙦𝙪𝙚𝙣𝙘𝙞𝙣𝙜, enabled by a 5′ RNA oligo ligation approach to recover missing 5′ sequence information 🧪 One of the most relevant findings is the functional link between analytical quality and biological performance. In the Cas9 mRNA/sgRNA model, increasing mRNA degradation was associated with a marked drop in knockout efficiency, reinforcing the importance of robust integrity monitoring during development and manufacturing. 📈 The work also shows strong alignment between nanopore results and orthogonal methods such as chromatography, mass spectrometry, Sanger sequencing, and capillary gel electrophoresis, while adding the advantage of single-molecule, multiattribute readout. 🎯 𝗞𝗲𝘆 𝘁𝗮𝗸𝗲-𝗮𝘄𝗮𝘆𝘀: • NDRS can combine 𝙞𝙙𝙚𝙣𝙩𝙞𝙩𝙮, 𝙞𝙣𝙩𝙚𝙜𝙧𝙞𝙩𝙮, 𝙘𝙖𝙥𝙥𝙞𝙣𝙜, 𝙖𝙣𝙙 𝙥𝙤𝙡𝙮(𝘼) 𝙖𝙣𝙖𝙡𝙮𝙨𝙞𝙨 in one platform • The method extends beyond mRNA to 𝙣𝙤𝙣𝙥𝙤𝙡𝙮𝙖𝙙𝙚𝙣𝙮𝙡𝙖𝙩𝙚𝙙 𝙨𝙜𝙍𝙉𝘼 • It provides 𝙛𝙪𝙣𝙘𝙩𝙞𝙤𝙣𝙖𝙡𝙡𝙮 𝙧𝙚𝙡𝙚𝙫𝙖𝙣𝙩 𝙦𝙪𝙖𝙡𝙞𝙩𝙮 𝙢𝙚𝙩𝙧𝙞𝙘𝙨 for RNA-based therapeutics • It has strong potential as a 𝙘𝙤𝙢𝙥𝙖𝙧𝙖𝙗𝙞𝙡𝙞𝙩𝙮 𝙖𝙣𝙙 𝙦𝙪𝙖𝙡𝙞𝙩𝙮-𝙘𝙤𝙣𝙩𝙧𝙤𝙡 𝙩𝙤𝙤𝙡 for next-generation RNA products #RNATherapeutics #mRNA #sgRNA #GeneEditing #NanoporeSequencing #AnalyticalScience #QualityControl #CellAndGeneTherapy Kamalakar Chatla, Brian Roper, Luladey Ayalew, Peggy Ko, Steffen Lippold, Meenakshi Doma, PhD & Julien Camperi

We need better tools to track RNA transcripts, in living cells and animals, across space and time. This new paper, which uses bioluminescence to pinpoint individual mRNAs, is a simple — yet elegant — way to do that. Historically, methods to track RNA in cells have relied on fluorescent probes. These probes grab onto mRNA and, when "excited" with external light, emit photons at a distinct, detectable wavelength. Excitation light, though, can cause lots of background noise or damage tissues. Light also doesn't penetrate deeply into the body! So you can't really use it to excite probes in an animal, and then get the signal back out again. This new paper gets around some of these problems by using a bioluminescent technology, called "RNA lanterns," to track transcripts without using any external light. It works in cell culture and in living mice. Here's how... First, the researchers split up a luciferase enzyme, called NanoLuc, into two halves. Each half was then fused to an RNA-binding protein — either MCP or PCP — which specifically bind to RNA aptamers, called MS2 and PP7, respectively. Next, the researchers encode these RNA aptamers into their target RNA molecule's sequence. When the RNA is transcribed, the NanoLuc halves basically 'spot' these aptamers and then grab onto them. When that happens, the two NanoLuc halves join together and a "pinprick" of light is emitted — bioluminescence. (In the GIF below, each dot is one mRNA transcript.) Using these RNA Lanterns, you can basically track a single type of RNA transcript in real-time (unfortunately, it can't be used to track hundreds of individual mRNAs in real-time and in living animals, which should be a future goal!) Previous papers have done similar things, but I think this is the first paper to do this using a single RNA aptamer, rather than a bunch of copies of them, to get a detectable signal. The researchers optimized the components a bunch; their RNA 'tag' is just 69 nucleotides in length, and they report a 330-fold increase over background compared to old versions of the technology. This system was tested in mammalian cells and live mice. For the latter, cells expressing the RNA lantern and tagged RNAs were implanted into the animals, and bioluminescent signals were detected within 30 seconds of transcription. Full paper: https://lnkd.in/ed8R6EEh

Nature Biomedical Engineering (31 Mar 2026) introduces "kinetic barcoding" — a novel multiplexed RNA detection strategy using CRISPR-Cas13a that identifies multiple viral targets simultaneously in a single droplet reaction, without requiring DNA amplification, separate fluorescence channels, or sample splitting. The authors discovered that LbuCas13a exhibits variable, crRNA/target-specific nuclease kinetics at the single-molecule level — not just differences in endpoint signal, but in the shape, slope, RMSD, and initiation time (T_init) of fluorescence trajectories. This variability was previously considered noise or a guide-screening nuisance; here it's exploited as a diagnostic signal. Prior to the kinetic barcoding paper, Cas13a diagnostics relied on one key mechanism: collateral (trans) cleavage — once Cas13a finds and binds its target RNA via CRISPR RNA (crRNA) that acts as the "address label" for CRISPR-Cas systems, it non-specifically chops up all nearby RNA molecules, including fluorescent reporter molecules. This "signal amplification" is what powers all prior diagnostic platforms. 👍 The igRNA (interfering gRNA) is a crRNA with a specific structural addition that changes how fast Cas13a works. The distinction is entirely novel to the kinetic barcoding paper; "igRNA" is a term coined by the authors themselves. This DNA tail is the key innovation. Because it is DNA (not RNA), Cas13a cannot cleave it — so it persists in the reaction and physically intrudes into Cas13a's HEPN active site, acting like a molecular speed bump. 🔅 Primary Patent: US20240309473A1 (https://lnkd.in/gaq7Faeq) — "KINETIC BARCODING TO ENHANCE SPECIFICITY OF CRISPR/CAS REACTIONS" Filed July 1, 2022 by Daniel A. Fletcher, Sungmin Son, Melanie Ott Key Claims: Covers the method of measuring/monitoring fluorescence kinetics of individual droplets containing target RNA + Cas RNP, including use of a RNA-polymer hybrid crRNA (the igRNA) where a polymer covalently linked to the 5′ end of the crRNA inhibits reporter cleavage 🔅 US20210348243A1 (https://lnkd.in/gDmQwybw) — "Rapid Field-Deployable Detection of SARS-CoV-2" Filed March 18, 2021 by Ott, Fozouni, Doudna, Fletcher, Son et al. — the foundational amplification-free droplet Cas13a SARS-CoV-2 detection method that preceded the kinetic barcoding work. Full Text: https://lnkd.in/g78quW78 #CRISPR #Cas13a #KineticBarcoding #CRISPRdiagnostics #igRNA #RNAdetection

"Spatial transcriptomics technologies with high resolution often lack high sensitivity in mRNA detection. Here we report a dendrimeric DNA coordinate barcoding design for spatial RNA sequencing (Decoder-seq), which offers both high sensitivity and high resolution. Decoder-seq combines dendrimeric nanosubstrates with microfluidic coordinate barcoding to generate spatial arrays with a DNA density approximately ten times higher than previously reported methods while maintaining flexibility in resolution. We show that the high RNA capture efficiency of Decoder-seq improved the detection of lowly expressed olfactory receptor (Olfr) genes in mouse olfactory bulbs and contributed to the discovery of a unique layer enrichment pattern for two Olfr genes. The near-cellular resolution provided by Decoder-seq has enabled the construction of a spatial single-cell atlas of the mouse hippocampus, revealing dendrite-enriched mRNAs in neurons. When applying Decoder-seq to human renal cell carcinomas, we dissected the heterogeneous tumor microenvironment across different cancer subtypes and identified spatial gradient-expressed genes related to epithelial–mesenchymal transition with the potential to predict tumor prognosis and progression." Interesting new spatial transcriptomics method by @Jiao Cao and larger team https://lnkd.in/eZDfxGkB

#SingleCell RNA sequencing of #SynovialFluid reveals dynamic immunoprofiles impacted by #TNF/#JAK inhibitors (adalimumab/tofacitinib). #KeyInsights: > Identification of #pathogenic cell types like SPP1+ macrophages and CXCL13+CD4+ T cells that are abundant in RA and reduced post-treatment. > Demonstration of drug-specific effects on gene expression, molecular pathways (e.g., JAK/STAT), and cell-cell communications. Tofacitinib showed a greater impact on diminishing cell-cell communications compared to adalimumab. > Highlighting the prolific communications between SPP1+/S100A12+ #macrophages and CXCL13+CD4+ T cells in RA, which decreased after treatment. These interactions may play a key role in RA pathogenesis and treatment outcomes. > The study suggests that synovial fluid analysis can reflect the pathogenic cell types and their communications in the synovial tissue, offering a less invasive approach for understanding RA. > Findings point towards a molecular basis for #PrecisionMedicine in RA, with potential implications for tailoring DMARD prescriptions. #ScienceCircuit #TranslationalResearch #SingleCell #PrecisionMedicine https://lnkd.in/erMxrxmp https://lnkd.in/e4khuq6x

#mRNA polyA tail is dynamic in cells. Viral sequences that recruit TENT4 to extend the poly(A) tail were found to enhance #mRNA stability. This paper showed results from screening 196,277 viral sequences which led to the discovery of five elements that are compatible with N1-methylpseudouridine. An element named A7 demonstrates particularly robust performance across cell types, delivery methods, modifications and coding sequences, making linear mRNA as stable as circular RNA while achieving higher translation efficiency. In mouse liver, A7-containing linear mRNA exhibits substantially higher protein levels than circular RNA, with sustained expression lasting for over 2 weeks. These RNA stability enhancers enable robust linear mRNA platforms that combine high and durable expression, low immunogenicity and simple manufacturing. https://lnkd.in/es2XmzQG

Can RNAseq replace the microscope? We explored this question by testing whether RNA sequencing can reliably mirror what pathologists see under the microscope using immunohistochemistry (IHC). Specifically, we examined how well RNA-seq detects clinically relevant cancer biomarkers across solid tumors and whether this approach would be diagnostically accurate. We analyzed expression levels of nine relevant biomarkers (ESR1, PGR, AR, MKI67, ERBB2, CD274, CDX2, KRT7, and KRT20) in 365 formalin-fixed, paraffin-embedded samples from breast, lung, gastrointestinal, and other solid tumors. By comparing detected transcripts per million with IHC scores, we derived RNAseq-based diagnostic thresholds that distinguish e.g., IHC-positive from IHC-negative cases. Our results showed strong correlations for most biomarkers (r=0.53-0.89), with RNA-seq thresholds achieving up to 98% diagnostic accuracy. Importanly, these findings held true in external (unseen) validation cohorts. We also noted that (a) tumor purity and (b) the tumor microenvironmental composition can act as confounders that affect the overall performance (e.g., moderate correlation for PD-L1 with r=0.63). So, to answer the question: can it replace the microscope? "Not yet but it can help". Our findings indicate that RNA-seq can serve as a robust objective, and scalable complement to IHC. The established thresholds enable reliable RNA based classification of biomarker status at scale. Since routine IHC is not scalable for screening hundreds of potential biomarkers, our findings provide a critical starting point for expanding transcriptomic approaches in clinical diagnostics (hence the "not yet"). Great teamwork by: Vladimir Kushnarev, Danil Stupichev, Suren Davitavyan, PhD, Kirill Kriukov, Basavaraja Uddajjara Shanthappa, Anna Butusova, Sofia Menshikova, Anna Belozerova, Anastasiia Shvyrkova, Arina Tkachuk, Olga Khatenkova, Linda Balabanian, Ekaterina Postovalova, Funda Meric-Bernstam and Alexander Bagaev link to publication in Scientific Reports: https://lnkd.in/eyBh77v6 pubmed: https://lnkd.in/ehkdwbBs Pubmed Central: https://lnkd.in/eVnXiTx6 #biomarker #RNAseq #NGS #IHC #digitalpathology #genomics #cancerresearch #diagnostics #precisiononcology #companiondiagnostics #laboratory #precisionmedicine #tumor BostonGene #breastcancer #coloncancer #NSCLC #lungcancer #PDL1 #immuotherapy #antibodydrugconjugates #adc #cancertherapy

I am pleased to share that our long-term collaborative research with Kyowa Kirin and Associate Professor Yasuaki Kimura at Nagoya University has been published in Nature Communications. In this work, we report the development of a fully chemically synthesized mRNA that exhibits over 100-fold higher translational output compared to canonical IVT mRNA, together with remarkably prolonged protein expression lasting more than 48 hours in cells—performance levels previously unattainable with enzymatically transcribed mRNA. Our study provides several key advances: First, by leveraging complete chemical synthesis, we established a platform enabling precise, position-specific introduction of ribose modifications within the open reading frame (ORF). This approach allowed us to systematically evaluate structural–activity relationships that were inaccessible with conventional IVT-based methods. Notably, we discovered that 2'-fluoro modification at the first nucleoside of each codon markedly stabilizes mRNA without impairing translation, overturning the long-standing assumption that ribose modification in ORFs is universally detrimental. Second, we found that extensive chemical modifications in the 5'-UTR can activate cap-independent translation with high efficiency. This enables robust protein synthesis even in the absence of a 5' cap structure—an important conceptual advance for the design of next-generation mRNA therapeutics. Third, by optimally combining ORF modifications with refined chemical designs at both the 5'-UTR and poly(A) tail, our chemically synthesized mRNA achieved higher and longer-lasting protein expression than state-of-the-art capped IVT mRNA (m1Ψ + extended poly(A)). In particular, the fully optimized construct (NK041) sustained high protein production for 24–48 hours and ultimately exhibited 6–16-fold higher expression than the best-performing IVT counterpart at later time points. These results demonstrate that rational chemical modification—made possible only through total chemical synthesis—provides a powerful and generalizable strategy for enhancing the stability and translational capacity of mRNA. We anticipate that this platform will contribute to future advances in cancer vaccines, protein replacement therapies, and regenerative medicine. This achievement would not have been possible without the dedicated efforts of our collaborators at Kyowa Kirin and the long-standing partnership with Associate Professor Kimura. I extend my sincere gratitude to all team members involved. Publication: Position-specific ORF nucleoside-ribose modifications enabled by complete chemical synthesis enhance mRNA stability and translation Nature Communications (2025) https://lnkd.in/gQqpHqNw

New tutorial on NGS101.com 🚀 Part 13 of my single-cell RNA-seq series is live: RNA Velocity Analysis with scVelo https://lnkd.in/gkTSa5qt Every analysis we've done so far — clustering, cell type annotation, trajectory, CellChat — captured a static snapshot of gene expression. RNA velocity goes one step further and tells you where each cell is headed. The secret? Unspliced pre-mRNA. When a gene is actively being turned on, unspliced RNA accumulates faster than it can be spliced. When a gene is shutting down, the opposite happens. By comparing spliced and unspliced counts for thousands of genes simultaneously, scVelo assigns each cell a velocity vector — a directional arrow showing its predicted future transcriptional state. In Part 13 you'll learn how to: → Generate spliced and unspliced count matrices with STARsolo → Transfer cell type labels and UMAP coordinates from your Seurat object into Python → Understand AnnData — Python's answer to the Seurat object → Fit scVelo's dynamical model to recover transcription, splicing, and degradation rates per gene → Visualize RNA velocity as directional stream plots on your UMAP → Compute latent time — a root-free, kinetics-based pseudotime → Identify genes with significantly different transcriptional kinetics between conditions This is also the first Python tutorial in the series. If you've only worked in R, don't worry — every step is explained from scratch and the biological context stays exactly the same. --- 🧪 Want to learn RNA-seq analysis hands-on? Cohort 1 of my Live RNA-seq Workshop for Absolute Beginners is now closed — but you can join the waitlist for the next cohort: https://lnkd.in/gXGEtBdH Spots fill fast. Get on the list early. #SingleCellRNAseq #RNAVelocity #scVelo #Bioinformatics #ComputationalBiology #NGS101 #scRNAseq #Python

Can nanopore sequencing revolutionize early disease detection? Current liquid biopsy techniques utilizing cell-free DNA often struggle to identify early-stage diseases due to their limited sensitivity. A recent groundbreaking study by Vikas Peddu, Karen Miga, Rebecca Fitzgerald, Daniel Kim, and their team from the University of California, Santa Cruz, and the University of Cambridge, published on bioRxiv, showcases the potential of long-read nanopore sequencing to be a game-changer. The researchers analyzed full-length cell-free RNA (cfRNA) from plasma samples to differentiate between healthy individuals, those with precancerous Barrett’s esophagus, and patients with esophageal adenocarcinoma. They discovered 270,679 novel intergenic cfRNAs and developed a tailored transcriptome reference for precise classification of both precancerous and cancerous conditions. Additionally, they pinpointed potential therapeutic targets within metabolic, signaling, and immune checkpoint pathways. These results highlight the efficacy of nanopore-based long-read RNA liquid biopsy platforms in early disease detection and targeted treatment, surpassing the capabilities of conventional methods. The methods developed in this study should be adaptable to detect other types of cancers using cfRNA in plasma. Exciting developments lie ahead in the realm of precision oncology! To delve deeper into the study, access the paper here: https://lnkd.in/erGvy_Wk #LiquidBiopsy #cfRNA #NanoporeSequencing #EarlyDetection #CancerDiagnostics #PrecisionMedicine #OncologyResearch