Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing
Open accessAbstract
CAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5′ end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries. Although these transcriptome profiling platforms have been demonstrated to be individually reproducible, no systematic comparison has been carried out between them. Here we compare CAGE, using both second- and third-generation sequencers, and RNA-seq, using a second-generation sequencer based on a panel of RNA mixtures from two human cell lines to examine power in the discrimination of biological states, detection of differentially expressed genes, linearity of measurements, and quantification reproducibility. We found that the quantified levels of gene expression are largely comparable across platforms and conclude that CAGE and RNA-seq are complementary technologies that can be used to improve incomplete gene models. We also found systematic bias in the second- and third-generation platforms, which is likely due to steps such as linker ligation, cleavage by restriction enzymes, and PCR amplification. This study provides a perspective on the performance of these platforms, which will be a baseline in the design of further experiments to tackle complex transcriptomes uncovered in a wide range of cell types.
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CAGE-seq determines the 5'-ends of TSSs at single nucleotide resolution and quantifies gene expression similar to RNA-seq.
“Cap Analysis of Gene Expression Sequencing (CAGE-seq) determines the 5’-ends of TSSs at single nucleotide resolution11,12 and quantifies gene expression similar to RNA-seq13.”
From Alternative promoters in CpG depleted regions are prevalently associated with epigenetic misregulation of liver cancer transcriptomes (2023) · cites this paper as [13]
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Cites
Landscape of transcription in human cells
Djebali, S., Davis, C. A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., et al. (2012) · Nature · doi:10.1038/nature11233 · Cited by 5,187 · see citing works
Self-citationGenome-wide analysis of mammalian promoter architecture and evolution
Carninci, P., Sandelin, A., Lenhard, B., Katayama, S., Shimokawa, K., Ponjavic, J., Semple, C. A. M., Taylor, M. S., Engström, P. G., Frith, M. C., et al. (2006) · Nature Genetics · doi:10.1038/ng1789 · Cited by 1,350 · see citing works
Self-citationNCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy
Pruitt, K. D., Tatusova, T., Brown, G. R., & Maglott, D. R. (2012) · Nucleic Acids Research · doi:10.1093/nar/gkr1079 · Cited by 1,149 · cited 2× · see citing works
“CAGE read alignments starting close to known TSSs represent our targeted signals; therefore, we examined the distance distribution of CAGE reads from known TSSs, which we defined as the 59 end of RefSeq transcripts (Pruitt et al. 2012), to determine the CAGE reads for gene expression analysis.”
BackgroundMethodStem cell transcriptome profiling via massive-scale mRNA sequencing
Cloonan, N., Forrest, A. R. R., Kolle, G., Gardiner, B. B. A., Faulkner, G. J., Brown, M. K., Taylor, D. F., Steptoe, A. L., Wani, S., Bethel, G., et al. (2008) · Nature Methods · doi:10.1038/nmeth.1223 · Cited by 1,035 · cited 3× · see citing works
“…of next-generation sequencing technologies has enabled us to identify and quantify transcripts well beyond previous microarray-based technologies (Cloonan et al. 2008; Marioni et al. 2008; Mortazavi et al. 2008; ‘t Hoen et al. 2008; Suzuki et al. 2009; Valen et al. 2009; Levin et al. 2010;…”
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Research works printed but not linked: 36
- Alon S, Vigneault F, Eminaga S, Christodoulou DC, Seidman JG, Church GM, Eisenberg E 2011 Barcoding bias in high-throughput multiplex sequencing of miRNA. Genome Res 21: 1506– 1511 21750102 PMC3166835
- Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, Itoh M, Kamiya M, Shibata K, Sasaki N, Izawa M, 1996 High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37: 327– 336 8938445
- Chen D, Patton JT 2001 Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5′-RACE and primer extension. Biotechniques 30: 574– 580, 582 11252793
- Dohm JC, Lottaz C, Borodina T, Himmelbauer H 2008 Substantial biases in ultra-short read data sets from high-throughput DNA sequencing. Nucleic Acids Res 36: e105 18660515 PMC2532726
- Du P, Kibbe WA, Lin SM 2008 lumi: a pipeline for processing Illumina microarray. Bioinformatics 24: 1547– 1548 18467348
- The ENCODE Project Consortium 2011 A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 9: e1001046 21526222 PMC3079585
- The ENCODE Project Consortium 2012 An integrated encyclopedia of DNA elements in the human genome. Nature 489: 57– 74 22955616 PMC3439153
- Forrest AR, Kawaji H, Rehli M, Baillie JK, de Hoon M, Haberle V, Lassmann T, Kulakovskiy IV, Lizio M, Itoh M, 2014 A promoter level mammalian expression atlas. Nature doi: 10.1038/nature13182 · DOI · Paper page
- Hadi SM, Bachi B, Shepherd JC, Yuan R, Ineichen K, Bickle TA 1979 DNA recognition and cleavage by the EcoP15 restriction endonuclease. J Mol Biol 134: 655– 666 231671
- Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Causey M, Colonell J, Dimeo J, Efcavitch JW, 2008 Single-molecule DNA sequencing of a viral genome. Science 320: 106– 109 18388294
- Itoh M, Kojima M, Nagao-Sato S, Saijo E, Lassmann T, Kanamori-Katayama M, Kaiho A, Lizio M, Kawaji H, Carninci P, 2012 Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer. PLoS ONE 7: e30809 22303458 PMC3268765
- Kanamori-Katayama M, Itoh M, Kawaji H, Lassmann T, Katayama S, Kojima M, Bertin N, Kaiho A, Ninomiya N, Daub CO, 2011 Unamplified cap analysis of gene expression on a single-molecule sequencer. Genome Res 21: 1150– 1159 21596820 PMC3129257
- Kawano M, Kawazu C, Lizio M, Kawaji H, Carninci P, Suzuki H, Hayashizaki Y 2010 Reduction of non-insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing. Biotechniques 49: 751– 755 20964636
- Kodzius R, Kojima M, Nishiyori H, Nakamura M, Fukuda S, Tagami M, Sasaki D, Imamura K, Kai C, Harbers M, 2006 CAGE: cap analysis of gene expression. Nat Methods 3: 211– 222 16489339
- Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ 2009 Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nat Methods 6: 291– 295 19287394 PMC2664327
- Kurosawa J, Nishiyori H, Hayashizaki Y 2011 Deep cap analysis of gene expression. Methods Mol Biol 687: 147– 163 20967606
- Lassmann T, Hayashizaki Y, Daub CO 2009 TagDust: a program to eliminate artifacts from next generation sequencing data. Bioinformatics 25: 2839– 2840 19737799 PMC2781754
- Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A, Regev A 2010 Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods 7: 709– 715 20711195 PMC3005310
- Li H, Durbin R 2009 Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754– 1760 19451168 PMC2705234
- Maeda N, Nishiyori H, Nakamura M, Kawazu C, Murata M, Sano H, Hayashida K, Fukuda S, Tagami M, Hasegawa A, 2008 Development of a DNA barcode tagging method for monitoring dynamic changes in gene expression by using an ultra high-throughput sequencer. Biotechniques 45: 95– 97 18611171
- Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y 2008 RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 18: 1509– 1517 18550803 PMC2527709
- Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B 2008 Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5: 621– 628 18516045 PMC13303166
- Nakamura K, Oshima T, Morimoto T, Ikeda S, Yoshikawa H, Shiwa Y, Ishikawa S, Linak MC, Hirai A, Takahashi H, 2011 Sequence-specific error profile of Illumina sequencers. Nucleic Acids Res 39: e90 21576222 PMC3141275
- Plessy C, Bertin N, Takahashi H, Simone R, Salimullah M, Lassmann T, Vitezic M, Severin J, Olivarius S, Lazarevic D, 2010 Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nat Methods 7: 528– 534 20543846 PMC2906222
- Ram O, Goren A, Amit I, Shoresh N, Yosef N, Ernst J, Kellis M, Gymrek M, Issner R, Coyne M, 2011 Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells. Cell 147: 1628– 1639 22196736 PMC3312319
- Robinson MD, McCarthy DJ, Smyth GK 2010 edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139– 140 19910308 PMC2796818
- Sam LT, Lipson D, Raz T, Cao X, Thompson J, Milos PM, Robinson D, Chinnaiyan AM, Kumar-Sinha C, Maher CA 2011 A comparison of single molecule and amplification based sequencing of cancer transcriptomes. PLoS ONE 6: e17305 21390249 PMC3046973
- Shen-Orr SS, Tibshirani R, Khatri P, Bodian DL, Staedtler F, Perry NM, Hastie T, Sarwal MM, Davis MM, Butte AJ 2010 Cell type-specific gene expression differences in complex tissues. Nat Methods 7: 287– 289 20208531 PMC3699332
- Shiraki T, Kondo S, Katayama S, Waki K, Kasukawa T, Kawaji H, Kodzius R, Watahiki A, Nakamura M, Arakawa T, 2003 Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc Natl Acad Sci 100: 15776– 15781 14663149 PMC307644
- Smyth GK 2004 Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3 16646809
- Suzuki R, Shimodaira H 2006 Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22: 1540– 1542 16595560
- Suzuki H, Forrest AR, van Nimwegen E, Daub CO, Balwierz PJ, Irvine KM, Lassmann T, Ravasi T, Hasegawa Y, de Hoon MJ, 2009 The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line. Nat Genet 41: 553– 562 19377474 PMC6711855
- Takahashi H, Lassmann T, Murata M, Carninci P 2012 5′ end-centered expression profiling using cap-analysis gene expression and next-generation sequencing. Nat Protoc 7: 542– 561 22362160 PMC4094379
- ’t Hoen PA, Ariyurek Y, Thygesen HH, Vreugdenhil E, Vossen RH, de Menezes RX, Boer JM, van Ommen GJ, den Dunnen JT 2008 Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms. Nucleic Acids Res 36: e141 18927111 PMC2588528
- Valen E, Pascarella G, Chalk A, Maeda N, Kojima M, Kawazu C, Murata M, Nishiyori H, Lazarevic D, Motti D, 2009 Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE. Genome Res 19: 255– 265 19074369 PMC2652207
- Wu ZJ, Meyer CA, Choudhury S, Shipitsin M, Maruyama R, Bessarabova M, Nikolskaya T, Sukumar S, Schwartzman A, Liu JS, 2010 Gene expression profiling of human breast tissue samples using SAGE-Seq. Genome Res 20: 1730– 1739 21045080 PMC2989999
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Li, B., Qing, T., Zhu, J., Wen, Z., Yu, Y., Fukumura, R., Zheng, Y., Gondo, Y., & Shi, L. (2017) · Scientific Reports · doi:10.1038/s41598-017-04520-z · Cited by 184 · see citing works
Nepal, C., & Andersen, J. B. (2023) · Nature Communications · doi:10.1038/s41467-023-38272-4 · Cited by 39 · see citing works
- Authors
- Kawaji, H., Lizio, M., Itoh, M., Kanamori-Katayama, M., Kaiho, A., Nishiyori-Sueki, H., Shin, J. W., Kojima-Ishiyama, M., Kawano, M., Murata, M., Ninomiya-Fukuda, N., Ishikawa-Kato, S., Nagao-Sato, S., Noma, S., Hayashizaki, Y., Forrest, A. R. R., Carninci, P., & The FANTOM Consortium
- Affiliations
- RIKEN (JP)
- Corresponding author
- Kawaji, Hideya
- Carninci, Piero
- ORCID
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2014-03-27
- Volume
- 24
- Issue
- 4
- Pages
- 708-717
- ISSN
- 1088-9051 (ISSN-L)
- PMC
- PMC3975069
- OpenAlex
- W2146975379
- Cited by
- 109
- References
- 4
- Citations by year
- 2026: 2 · 2025: 5 · 2024: 3 · 2023: 14 · 2022: 8 · 2021: 10 · 2020: 9 · 2019: 12 · 2018: 5 · 2017: 11 · 2016: 15 · 2015: 11 · 2014: 4
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genome Research (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UWA Profiles and Research Repository (UWA) (submittedVersion, cc-by)
- HighWire Press Open Archive (submittedVersion)
- Funders
- Ministry of Education, Culture, Sports, Science and Technology
- RIKEN
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
- Keywords
- CAGE, RNA-seq, gene expression profiling, platform comparison, single-molecule sequencing, PCR bias, gene expression quantification, differentially expressed genes, measurement reproducibility, human cell lines, gene models
- MeSH terms
- Humans, RNA, Sequence Analysis, RNA, Gene Expression Profiling, High-Throughput Nucleotide Sequencing, Transcriptome
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