Menu

Close

Template switching causes artificial junction formation and false identification of circular RNAs

Chong Tang, Tian Yu, Yeming Xie, Zhuqing Wang, Hayden McSwiggin, Ying Zhang, Huili Zheng, Wei Yan

Preprint posted on February 06, 2018 https://www.biorxiv.org/content/early/2018/02/06/259556

Magic circles: the illusion of circular RNAs created by template-switching of MMLV-derived reverse transcriptases during RNA-Seq library preparation

Selected by Fabio Liberante

 

The preprint and its context

The study of circular RNAs has grown massively in the last decade or so, not least because of their increasing association with a huge array of both developmental and disease-related processes in cells. As demand to profile and understand these mysterious RNAs grows, so does the demand for pipelines to analyse the huge amount of RNA-Seq data that the scientific community continues to generate. However, such a rapid expansion of tools and techniques can often be at risk of overlooking caveats in the subtleties of the molecular processes used at the lab bench.

This preprint has highlighted such a caveat in using standard MMLV-derived reverse transcriptases (RT) to create cDNA libraries for RNA-Seq. The template-switching activity of these enzymes means that they add a small number of extra nucleotides at the 3’ end of transcripts. These can then anneal to a different part of the same RNA template and continue extending the transcript. The created transcript now contains junctions that were absent from the original RNA pool and which could be identified as circRNAs by bioinformatics analysis.

 

MMLV enzyme creating artificial junctions and "circRNAs"

Figure 1A from the preprint: Schematic of the mechanism through which the terminal transferase activity of MMLV-derived reverse transcriptase can add several non-templated nucleotides to the 3’ end of cDNA. Template-switching can then generate artificial junctions derived from linear RNA templates.

 

The authors examined whether detection of circular RNAs in samples might be an artefact of the reverse transcriptase used during cDNA synthesis. This hypothesis was tested through a number of different experiments, but the simplest one generated synthetic circular RNAs from linear ones using T4 RNA ligase and used these as positive and negative controls, respectively, in standard cDNA protocols. They indeed found that using an RT enzyme typically provided with RNA-Seq kits created these artificial junctions from linear RNAs, which could be detected by divergent primers, as illustrated in the figure above.

The authors also demonstrated that RNase R treatment before cDNA synthesis is not sufficient to remove all linear RNAs and can actually degrade circRNAs, likely through hydrolysis. Worryingly, this is something many RNA-Seq protocols have begun to include to enrich for circRNAs.

If these findings hold up under scrutiny by other groups, this could be a not insignificant setback for circRNA research. All is not lost, however. While they showed that their methodology identified considerably fewer “circRNAs” than a previous comparable study, the ones that were identified showed significant overlap, suggesting that the true positive results in existing studies are still there to be found, despite the artefacts that the datasets contain.

 

What makes this study interesting

This preprint initially caught my attention because “false identification of circular RNAs” is quite an alarming phrase to read in a title. Unfortunately, studies that highlight flaws in widely used protocols can still be difficult to sell convincingly to editors. This makes it an excellent example of the benefits of preprint publishing; it made the findings public quickly so that others don’t fall victim to the same problem whilst the paper undergoes the lengthy review and editorial process.

Part of its usefulness is in its simplicity – it’s only 4 figures. The authors, demonstrate the problem with a simple artificial template, then commonly-employed RNA spike-ins and finally a set of real-world samples. Finally, like any form of criticism, it is best when constructive. The authors offer a very simple solution to the problem: swap RT enzyme. They also offer a new method of validating identified circRNAs using High-resolution melting (HRM) analysis, something that could be implemented in most labs.

 

Questions and future directions

It would be interesting to know what alerted the authors to the issue of false junctions. Was it an unexpected result or was it based on their insight into the biochemistry of virus-derived reverse transcriptases?

The authors only describe MonsterScript as a suitable enzyme that can be easily substituted to solve the issue they identified. However, some cursory research seems to suggest that this product is no longer available. It would have been helpful if the authors provided more detail or speculation on which domains of this enzyme were likely modified by the supplier to eliminate the template-switching activity. This would allow other alternatives to be easily identified. Even more useful, they could have tested a number of other RT enzymes and perhaps even the more modern non-MMLV derived types, such as TGIRT enzymes or the recently published MarathonRT.

Further, the authors only used a single suite of tools, namely bwa and CIRI2, to analyse the generated RNA-Seq data. It would have been interesting to compare how other tools handled or filtered the low junction ratios that the authors say are usually ascribed to false junctions.

 

Further reading

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron
RNA. 2018 Feb;24(2):183-195. doi: 10.1261/rna.063479.117
Zhao C, Liu F, Pyle AM
https://www.ncbi.nlm.nih.gov/pubmed/29109157

Emerging roles and context of circular RNAs
Wiley Interdiscip Rev RNA. 2017 Mar;8(2). doi: 10.1002/wrna.1386
Panda AC, Grammatikakis I, Munk R, Gorospe M, Abdelmohsen K
https://www.ncbi.nlm.nih.gov/pubmed/27612318

Tags: circrna, circrnas, circular rna, hrm, mmlv, monsterscript, rna-seq, rt, superscript

Posted on: 23rd July 2018 , updated on: 24th July 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the biochemistry category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

    Keith T Woodley, Mark O Collins



    Selected by Abagael Lasseigne

    3

    A complex containing lysine-acetylated actin inhibits the formin INF2

    Mu A, Tak Shun Fung, Arminja N. Kettenbach, et al.



    Selected by Laura McCormick

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Activation of intracellular transport by relieving KIF1C autoinhibition

    Nida Siddiqui, Alice Bachmann, Alexander J Zwetsloot, et al.



    Selected by Ben Craske, Thibault Legal and Toni McHugh

    1

    Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis

    Jonathan P Gumucio, Austin H Qasawa, Patrick J Ferrara, et al.



    Selected by Pablo Ranea Robles

    Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

    Diego Gauto, Leandro Estrozi, Charles Schwieters, et al.



    Selected by Reid Alderson

    1

    Disrupting Transcriptional Feedback Yields an Escape-Resistant Antiviral

    Sonali Chaturvedi, Marie Wolf, Noam Vardi, et al.



    Selected by Pavithran Ravindran

    1

    Structure of a cytochrome-based bacterial nanowire

    David J Filman, Stephen F Marino, Joy E Ward, et al.



    Selected by Amberley Stephens

    Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

    Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, et al.

    AND

    Live-cell single RNA imaging reveals bursts of translational frameshifting

    Kenneth R Lyon Jr, Luis U Aguilera, Tatsuya Morisaki, et al.



    Selected by Nicola Stevenson

    Retrieving High-Resolution Information from Disordered 2D Crystals by Single Particle Cryo-EM

    Ricardo Righetto, Nikhil Biyani, Julia Kowal, et al.



    Selected by David Wright

    Structural venomics: evolution of a complex chemical arsenal by massive duplication and neofunctionalization of a single ancestral fold

    Sandy Steffany Pineda, Yanni K-Y Chin, Eivind A.B. Undheim, et al.



    Selected by Tessa Sinnige

    Also in the bioinformatics category:

    Lineage tracing on transcriptional landscapes links state to fate during differentiation

    Caleb Weinreb, Alejo E Rodriguez-Fraticelli, Fernando D Camargo, et al.



    Selected by Yen-Chung Chen

    1

    Charting a tissue from single-cell transcriptomes

    Mor Nitzan, Nikos Karaiskos, Nir Friedman, et al.



    Selected by Irepan Salvador-Martinez

    Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions

    Hila Sberro, Nicholas Greenfield, Georgios Pavlopoulos, et al.



    Selected by Ganesh Kadamur

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    The embryonic transcriptome of Arabidopsis thaliana

    Falko Hofmann, Michael A Schon, Michael D Nodine



    Selected by Chandra Shekhar Misra

    1

    The landscape of antigen-specific T cells in human cancers

    Bo Li, Longchao Liu, Jian Zhang, et al.



    Selected by Rob Hynds

    1

    Single-cell RNA sequencing reveals novel cell differentiation dynamics during human airway epithelium regeneration

    Sandra Ruiz Garcia, Marie Deprez, Kevin Lebrigand, et al.



    Selected by Rob Hynds

    1

    PUMILIO hyperactivity drives premature aging of Norad-deficient mice

    Florian Kopp, Mehmet Yalvac, Beibei Chen, et al.



    Selected by Carmen Adriaens

    Target-specific precision of CRISPR-mediated genome editing

    Anob M Chakrabarti, Tristan Henser-Brownhill, Josep Monserrat, et al.



    Selected by Rob Hynds

    1

    Precise tuning of gene expression output levels in mammalian cells

    Yale S. Michaels, Mike B Barnkob, Hector Barbosa, et al.



    Selected by Tim Fessenden

    1

    Template switching causes artificial junction formation and false identification of circular RNAs

    Chong Tang, Tian Yu, Yeming Xie, et al.



    Selected by Fabio Liberante

    An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics

    Ilias Angelidis, Lukas M Simon, Isis E Fernandez, et al.



    Selected by Rob Hynds

    1

    SWI/SNF remains localized to chromatin in the presence of SCHLAP1

    Jesse R Raab, Keriayn N Smith, Camarie C Spear, et al.



    Selected by Carmen Adriaens

    1

    Atomic model of microtubule-bound tau

    Elizabeth H Kellogg, Nisreen M.A. Hejab, Simon Poepsel, et al.



    Selected by Satish Bodakuntla

    1

    Higher-Order Organization Principles of Pre-translational mRNPs

    Mihir Metkar, Hakan Ozadam, Bryan R. Lajoie, et al.



    Selected by Carmen Adriaens

    Also in the cell biology category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Minimal membrane interactions conferred by Rheb C-terminal farnesylation are essential for mTORC1 activation

    Shawn M Ferguson, Brittany Angarola



    Selected by Sandra Malmgren Hill

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Vibha SINGH

    EHD2-mediated restriction of caveolar dynamics regulates cellular lipid uptake

    Claudia Matthaeus, Ines Lahmann, Severine Kunz, et al.



    Selected by Andreas Müller

    1

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Joseph Jose Thottacherry

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    A metabolic switch from OXPHOS to glycolysis is essential for cardiomyocyte proliferation in the regenerating heart

    Hessel Honkoop, Dennis de Bakker, Alla Aharonov, et al.



    Selected by Andreas van Impel

    1

    S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

    Keith T Woodley, Mark O Collins



    Selected by Abagael Lasseigne

    3

    A complex containing lysine-acetylated actin inhibits the formin INF2

    Mu A, Tak Shun Fung, Arminja N. Kettenbach, et al.



    Selected by Laura McCormick

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    1

    Actomyosin-II facilitates long-range retrograde transport of large cargoes by controlling axonal radial contractility

    Tong Wang, Wei Li, Sally Martin, et al.



    Selected by Ivana Viktorinová

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Also in the genomics category:

    High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue-specificity

    Kaia Mattioli, Pieter-Jan Volders, Chiara Gerhardinger, et al.



    Selected by Clarice Hong

    Reconstruction of the global neural crest gene regulatory network in vivo

    Ruth M Williams, Ivan Candido-Ferreira, Emmanouela Repapi, et al.



    Selected by Hannah Brunsdon

    Charting a tissue from single-cell transcriptomes

    Mor Nitzan, Nikos Karaiskos, Nir Friedman, et al.



    Selected by Irepan Salvador-Martinez

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    1

    Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

    Julien Rougot, Naomi D Chrispijn, Marco Aben, et al.



    Selected by Yen-Chung Chen

    1

    The embryonic transcriptome of Arabidopsis thaliana

    Falko Hofmann, Michael A Schon, Michael D Nodine



    Selected by Chandra Shekhar Misra

    1

    Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells

    Mridusmita Saikia, Philip Burnham, Sara H Keshavjee, et al.

    AND

    High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

    Mandeep Singh, Ghamdan Al-Eryani, Shaun Carswell, et al.



    Selected by Samantha Seah

    The microbial basis of impaired wound healing: differential roles for pathogens, "bystanders", and strain-level diversification in clinical outcomes

    Lindsay Kalan, Jacquelyn S Meisel, Michael A Loesche, et al.



    Selected by Snehal Kadam

    Comparative analysis of droplet-based ultra-high-throughput single-cell RNA-seq systems

    Xiannian Zhang, Tianqi Li, Feng Liu, et al.



    Selected by Samantha Seah

    PUMILIO hyperactivity drives premature aging of Norad-deficient mice

    Florian Kopp, Mehmet Yalvac, Beibei Chen, et al.



    Selected by Carmen Adriaens

    LCM-seq reveals unique transcriptional adaption mechanisms of resistant neurons in spinal muscular atrophy

    Susanne Nichterwitz, Helena Storvall, Jik Nijssen, et al.

    AND

    Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS

    Jik Nijssen, Julio Cesar Aguila Benitez, Rein Hoogstraaten, et al.



    Selected by Yen-Chung Chen

    LADL: Light-activated dynamic looping for endogenous gene expression control

    Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, et al.



    Selected by Ivan Candido-Ferreira

    Precise tuning of gene expression output levels in mammalian cells

    Yale S. Michaels, Mike B Barnkob, Hector Barbosa, et al.



    Selected by Tim Fessenden

    1

    Template switching causes artificial junction formation and false identification of circular RNAs

    Chong Tang, Tian Yu, Yeming Xie, et al.



    Selected by Fabio Liberante

    The genomic basis of colour pattern polymorphism in the harlequin ladybird

    Mathieu Gautier, Junichi Yamaguchi, Julien Foucaud, et al.



    Selected by Fillip Port

    Widespread inter-individual gene expression variability in Arabidopsis thaliana

    Sandra Cortijo, Zeynep Aydin, Sebastian Ahnert, et al.



    Selected by Martin Balcerowicz

    Also in the molecular biology category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

    Anushila Chatterjee, Cydney N Johnson, Phat Luong, et al.



    Selected by Yasmin Lau

    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.



    Selected by Srivats Venkataramanan

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Unlimited genetic switches for cell-type specific manipulation

    Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, et al.



    Selected by Rafael Almeida

    1

    Stable knockout and complementation of receptor expression using in vitro cell line derived reticulocytes for dissection of host malaria invasion requirements

    Timothy J Satchwell, Katherine E Wright, Katy L Haydn-Smith, et al.



    Selected by Alyson Smith

    The coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

    Yiqin Ma, Daniel J McKay, Laura Buttitta



    Selected by Gabriel Aughey

    1

    Disrupting Transcriptional Feedback Yields an Escape-Resistant Antiviral

    Sonali Chaturvedi, Marie Wolf, Noam Vardi, et al.



    Selected by Pavithran Ravindran

    1

    Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

    Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, et al.



    Selected by Lorenzo Lafranchi
    Close