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RNA sequencing

RNA sequencing

RNA sequencing techniques are used to determine the sequence of nucleotide bases, adenine (A), cytosine (C), guanine (G) and uracil (U) in RNA molecules.

Usually RNA is first converted into cDNA (complementary DNA) with a reverse transcriptase enzyme, and then a second strand synthesis reaction is performed so DNA sequencing techniques can be applied to double stranded DNA copies of RNA transcripts. Since this method can lose information from the 5' and 3' ends of the transcript, other methods have been developed that omit the second strand synthesis reaction and ligate adapters to the cDNA for sequencing reactions. Sequencing adapters are consistent sequences that, when applied to flank the cDNA fragments, produce a cDNA library.

RNA sequencing is replacing gene expression arrays to analyze the spectrum and abundance of transcripts in a given cell or tissue type at a given time. The technique called RNA-Seq, also known as whole transcriptome shotgun sequencing, generates cDNA and uses it in next-generation sequencing.

UK-based Oxford Nanopore Technologies devised a system to directly sequence RNA with a device called the MinION, in which electrical current is applied across a nanoscale molecular pore and current fluctuations detect the RNA sequence as the RNA molecule snakes through the pore. This RNA sequencing device was used by NASA on the International Space Station because NASA is interested in using it to identify onboard microorganisms and to monitor changes in human health or microbiomes; NASA is also interested in the possibility of detecting life based on DNA or RNA elsewhere in the universe.

RNA-Seq delivers an unbiased and unprecedented high-resolution view of the global transcriptional landscape, which allows an affordable and accurate approach for gene expression quantification and differential gene expression analysis between multiple groups of samples. RNA-Seq can identify novel and previously unexpected transcripts without the need for a reference genome, allowing de novo assembly of new transcriptome that is not previously studied before. It also enables the discovery of novel gene structures, alternatively spliced isoforms, gene fusions, SNPs/InDel, and allele-specific expression (ASE).RNA-Seq is a sensitive tool for gene expression profiling. Compared to microarray, RNA-Seq offers a digital read that is more accurate for all gene expression.



Further reading

Documentaries, videos and podcasts



Mark Terry
May 14, 2021
Every week there are numerous scientific studies published. Here's a look at some of the more interesting ones.
Ricard Argelaguet
May 3, 2021
Nature Biotechnology
The development of single-cell multimodal assays provides a powerful tool for investigating multiple dimensions of cellular heterogeneity, enabling new insights into development, tissue homeostasis and disease. A key challenge in the analysis of single-cell multimodal data is to devise appropriate strategies for tying together data across different modalities. The term 'data integration' has been used to describe this task, encompassing a broad collection of approaches ranging from batch correction of individual omics datasets to association of chromatin accessibility and genetic variation with transcription. Although existing integration strategies exploit similar mathematical ideas, they typically have distinct goals and rely on different principles and assumptions. Consequently, new definitions and concepts are needed to contextualize existing methods and to enable development of new methods. As the number of single-cell experiments with multiple data modalities increases, Argelaguet and colleagues review the concepts and challenges of data integration.
Science X staff
May 3, 2021
Researchers at the University of Haifa, the Weizmann Institute and the Center for Genomic Regulation (CRG) have built the first atlas of all of the different types of cells in Stylophora pistillata, a reef-building stony coral native to the Indo-Pacific oceans. Published today in the journal Cell, the study is the first to detect the presence of specialized immune cells in corals.
Akira Cortal
April 29, 2021
Nature Biotechnology
Because of the stochasticity associated with high-throughput single-cell sequencing, current methods for exploring cell-type diversity rely on clustering-based computational approaches in which heterogeneity is characterized at cell subpopulation rather than at full single-cell resolution. Here we present Cell-ID, a clustering-free multivariate statistical method for the robust extraction of per-cell gene signatures from single-cell sequencing data. We applied Cell-ID to data from multiple human and mouse samples, including blood cells, pancreatic islets and airway, intestinal and olfactory epithelium, as well as to comprehensive mouse cell atlas datasets. We demonstrate that Cell-ID signatures are reproducible across different donors, tissues of origin, species and single-cell omics technologies, and can be used for automatic cell-type annotation and cell matching across datasets. Cell-ID improves biological interpretation at individual cell level, enabling discovery of previously uncharacterized rare cell types or cell states. Cell-ID is distributed as an open-source R software package. Cell-ID facilitates the analysis of cell-type heterogeneity and cell identity across multiple samples at the single-cell level.
Jérémie Breda
April 29, 2021
Nature Biotechnology
Despite substantial progress in single-cell RNA-seq (scRNA-seq) data analysis methods, there is still little agreement on how to best normalize such data. Starting from the basic requirements that inferred expression states should correct for both biological and measurement sampling noise and that changes in expression should be measured in terms of fold changes, we here derive a Bayesian normalization procedure called Sanity (SAmpling-Noise-corrected Inference of Transcription activitY) from first principles. Sanity estimates expression values and associated error bars directly from raw unique molecular identifier (UMI) counts without any tunable parameters. Using simulated and real scRNA-seq datasets, we show that Sanity outperforms other normalization methods on downstream tasks, such as finding nearest-neighbor cells and clustering cells into subtypes. Moreover, we show that by systematically overestimating the expression variability of genes with low expression and by introducing spurious correlations through mapping the data to a lower-dimensional representation, other methods yield severely distorted pictures of the data. A Bayesian procedure overcomes challenges in single-cell RNA-seq data normalization.
Brenda Wingfield
April 21, 2021
In the past year the world has been overwhelmed with rapidly emerging, important and fascinating information regarding SARS CoV-2, the virus that causes COVID-19. The pace of learning has been astounding, not just for the general public but for virus experts.
Halima Hannah Schede
April 19, 2021
Nature Biotechnology
Several techniques are currently being developed for spatially resolved omics profiling, but each new method requires the setup of specific detection strategies or specialized instrumentation. Here we describe an imaging-free framework to localize high-throughput readouts within a tissue by cutting the sample into thin strips in a way that allows subsequent image reconstruction. We implemented this framework to transform a low-input RNA sequencing protocol into an imaging-free spatial transcriptomics technique (called STRP-seq) and validated it by profiling the spatial transcriptome of the mouse brain. We applied the technique to the brain of the Australian bearded dragon, Pogona vitticeps. Our results reveal the molecular anatomy of the telencephalon of this lizard, providing evidence for a marked regionalization of the reptilian pallium and subpallium. We expect that STRP-seq can be used to derive spatially resolved data from a range of other omics techniques. A refined spatial sampling technique transforms standard RNA sequencing into a spatial transcriptomics method.
Chao Gao
April 19, 2021
Nature Biotechnology
Integrating large single-cell gene expression, chromatin accessibility and DNA methylation datasets requires general and scalable computational approaches. Here we describe online integrative non-negative matrix factorization (iNMF), an algorithm for integrating large, diverse and continually arriving single-cell datasets. Our approach scales to arbitrarily large numbers of cells using fixed memory, iteratively incorporates new datasets as they are generated and allows many users to simultaneously analyze a single copy of a large dataset by streaming it over the internet. Iterative data addition can also be used to map new data to a reference dataset. Comparisons with previous methods indicate that the improvements in efficiency do not sacrifice dataset alignment and cluster preservation performance. We demonstrate the effectiveness of online iNMF by integrating more than 1 million cells on a standard laptop, integrating large single-cell RNA sequencing and spatial transcriptomic datasets, and iteratively constructing a single-cell multi-omic atlas of the mouse motor cortex. A new algorithm enables scalable and iterative integration of single-cell datasets.
Bio-Techne Corporation
April 15, 2021
/PRNewswire/ -- Bio-Techne Corporation (NASDAQ: TECH) today announced that Advanced Cell Diagnostics (ACD), a Bio-Techne brand, completed an extensive...
Páll Melsted
April 1, 2021
Nature Biotechnology
We describe a workflow for preprocessing of single-cell RNA-sequencing data that balances efficiency and accuracy. Our workflow is based on the kallisto and bustools programs, and is near optimal in speed with a constant memory requirement providing scalability for arbitrarily large datasets. The workflow is modular, and we demonstrate its flexibility by showing how it can be used for RNA velocity analyses. A preprocessing workflow for single-cell RNA-seq data achieves near-optimal speed.
Zymo Research Corp.
February 11, 2021
/PRNewswire/ -- Zymo Research announced today their COVID-19 Variant Sequencing Service. This new global service expands Zymo Research's end-to-end SARS-CoV-2...
Research and Markets
December 21, 2020
/PRNewswire/ -- The "The Market for RNA Sequencing" report has been added to's offering. The sequencing market has continued to provide...
Science X staff
December 21, 2020
We've all suffered from viruses, but did you know that they are also a problem for mushrooms and molds? Mycoviruses are viruses that specifically infect fungi and have the potential to impact ecology, agriculture, food security, and public health. Understanding the nature of these viruses, including their number and evolution, can help us understand their origins and inform our understanding of viruses in general.
Research and Markets
November 26, 2020
/PRNewswire/ -- The "RNA Analysis/Transcriptomics Market by Product (Reagents, Instruments, Software), Technology (Microarrays, NGS, Sanger), Application...
Zhang Nannan
November 23, 2020
After double fertilization, zygotic activation occurs that initiates a new life cycle, followed by cell divisions, cell differentiation and organogenesis. During post-embryonic development, stem cells located in shoot apical meristem (SAM) and root apical meristem (RAM) allow plants to continuously generate new tissues and organs. Therefore, understanding the role of zygote activation and stem cell homeostasis is a long-standing interest to scientists.
Science X staff
November 18, 2020
Organ and stem cell transplants are proven and frequently used methods in modern clinical practice. However, even when performed regularly in specialized centers, some patients still experience a number of serious complications afterwards. Among other things, infections with fungi and viruses can jeopardize therapeutic success. For example, coinfection with cytomegalovirus, which belongs to the family of Herpes viruses, and the fungus Aspergillus fumigatus can be critical. This combination of pathogens poses a serious medical threat in organ and stem cell transplantation.
November 17, 2020
NantHealth and ImmunityBio Announce RNA Profiling for Clinical Decision Support Publication In Nature's Scientific Reports - read this article along with other careers information, tips and advice on BioSpace
Hospital for Special Surgery
November 8, 2020
/PRNewswire/ -- Since doctors began treating cancer patients with immunotherapy drugs called checkpoint inhibitors nearly a decade ago, they have observed that...
Esther Landhuis
July 28, 2020
Scientific American
Scientific American is the essential guide to the most awe-inspiring advances in science and technology, explaining how they change our understanding of the world and shape our lives.


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