Biology:Cap analysis of gene expression

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Cap analysis of gene expression (CAGE) is a gene expression technique used in molecular biology to produce a snapshot of the 5′ end of the messenger RNA population in a biological sample (the transcriptome). The small fragments (historically 27 nucleotides long, but now limited only by sequencing technologies) from the very beginnings of mRNAs (5' ends of capped transcripts) are extracted, reverse-transcribed to cDNA, PCR amplified (if needed) and sequenced. CAGE was first published by Hayashizaki, Carninci and co-workers in 2003.[1] CAGE has been extensively used within the FANTOM research projects.

Analysis

The output of CAGE is a set of short nucleotide sequences (often called tags in analogy to expressed sequence tags) with their observed counts. Copy numbers of CAGE tags provide a digital quantification of the RNA transcript abundances in biological samples. Using a reference genome, a researcher can usually determine, with some confidence, the original mRNA (and therefore which gene) the tag was extracted from.

Unlike a similar technique serial analysis of gene expression (SAGE) in which tags come from other parts of transcripts, CAGE is primarily used to locate exact transcription start sites in the genome. This knowledge in turn allows a researcher to investigate promoter structure necessary for gene expression.

CAGE tags tend to start with an extra guanine (G) that is not encoded in the genome, which is attributed to the template-free 5′-extension during the first-strand cDNA synthesis[2] or reverse-transcription of the cap itself.[3] When not corrected, this can induce erroneous mapping of CAGE tags, for instance to nontranscribed pseudogenes.[2] On the other hand, this addition of Gs was also utilised as a signal to filter more reliable TSS peaks.[4]

History

The original CAGE method (Shiraki et al., 2003)[1] was using CAP Trapper[5] for capturing the 5′ ends, oligo-dT primers for synthesizing the cDNAs, the type IIs restriction enzyme MmeI for cleaving the tags, and the Sanger method for sequencing them.

Random reverse-transcription primers were introduced in 2006 by Kodzius et al.[6] to better detect the non-polyadenylated RNAs.

In DeepCAGE (Valen et al., 2008),[7] the tag concatemers were sequenced at a higher throughput on the 454 “next-generation” sequencing platform.

In 2008, barcode multiplexing was added to the DeepCAGE protocol (Maeda et al., 2008).[8]

In nanoCAGE (Plessy et al., 2010),[9] the 5′ ends or RNAs were captured with the template-switching method instead of CAP Trapper, in order to analyze smaller starting amounts of total RNA. Longer tags were cleaved with the type III restriction enzyme EcoP15I and directly sequenced on the Solexa (then Illumina) platform without concatenation.

The CAGEscan methodology (Plessy et al., 2010),[9] where the enzymatic tag cleavage is skipped, and the 5′ cDNAs sequenced paired-end, was introduced in the same article to connect novel promoters to known annotations.

With HeliScopeCAGE (Kanamori-Katayama et al., 2011),[10] the CAP-trapped CAGE protocol was changed to skip the enzymatic tag cleavage and sequence directly the capped 5′ ends on the HeliScope platform, without PCR amplification. It was then automated by Itoh et al.[11] in 2012.

In 2012, the standard CAGE protocol was updated by Takahashi et al.[12] to cleave tags with EcoP15I and sequence them on the Illumina-Solexa platform.

In 2013, Batut et al.[13] combined CAP trapper, template switching, and 5′-phosphate-dependent exonuclease digestion in RAMPAGE to maximize promoter specificity.

In 2014, Murata et al.[14] published the nAnTi-CAGE protocol, where capped 5′ ends are sequenced on the Illumina platform with no PCR amplification and no tag cleavage.

In 2017, Poulain et al.[15] updated the nanoCAGE protocol to use the tagmentation method (based on Tn5 transposition) for multiplexing.

In 2018, Cvetesic et al.[16] increased the sensitivity of CAP-trapped CAGE by introducing selectively degradable carrier RNA (SLIC-CAGE, "Super-Low Input Carrier-CAGE").

In 2021, Takahashi et al.[17] simplified the sequencing of CAGE libraries on Illumina sequencers by skipping second-strand synthesis directly loading single-strand cDNAs (Low Quantity Single Strand CAGE, "LQ-ssCAGE").

See also

References

  1. ↑ 1.0 1.1 Shiraki, TExpression error: Unrecognized word "et". (2003-12-23). "Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage". Proc Natl Acad Sci U S A 100 (26): 15776–81. doi:10.1073/pnas.2136655100. PMID 14663149. Bibcode: 2003PNAS..10015776S. 
  2. ↑ 2.0 2.1 Zhao, Xiaobei (2011). "Systematic Clustering of Transcription Start Site Landscapes". PLOS ONE 6 (8): e23409. doi:10.1371/journal.pone.0023409. PMID 21887249. Bibcode: 2011PLoSO...623409Z. 
  3. ↑ Ohtake, Hideki; Ohkoto, Kuniyo; Ishimaru, Yoshihiro; Kato, Seishi (2004). "Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method". DNA Res. 11 (4): 305–9. doi:10.1093/dnares/11.4.305. PMID 15500255. 
  4. ↑ Cumbie, Jason (2015). "NanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites". BMC Genomics 16 (1): 597. doi:10.1186/s12864-015-1670-6. PMID 26268438. 
  5. ↑ Carninci, Piero (1996). "High-efficiency full-length cDNA cloning by biotinylated CAP trapper.". Genomics 37 (3): 327–36. doi:10.1006/geno.1996.0567. PMID 8938445. 
  6. ↑ Kodzius, Rimantas (2006). "CAGE: cap analysis of gene expression.". Nat Methods 3 (3): 211–22. doi:10.1038/nmeth0306-211. PMID 16489339. 
  7. ↑ Valen, Eivind (2009). "Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE.". Genome Res. 19 (2): 255–265. doi:10.1101/gr.084541.108. PMID 19074369. 
  8. ↑ Maeda, Norihiro (2008). "Development of a DNA barcode tagging method for monitoring dynamic changes in gene expression by using an ultra high-throughput sequencer.". BioTechniques 45 (1): 95–7. doi:10.2144/000112814. PMID 18611171. http://www.biotechniques.com/article/000112814. Retrieved 2016-04-28. 
  9. ↑ 9.0 9.1 Plessy, Charles (2010). "Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan". Nat Methods 7 (7): 528–34. doi:10.1038/nmeth.1470. PMID 20543846. 
  10. ↑ Kanamori-Katayama, Mutsumi (2011). "Unamplified cap analysis of gene expression on a single-molecule sequencer.". Genome Res. 21 (7): 1150–9. doi:10.1101/gr.115469.110. PMID 21596820. 
  11. ↑ Itoh, Masayoshi (2012). "Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.". PLOS ONE 7 (1): e30809. doi:10.1371/journal.pone.0030809. PMID 22303458. Bibcode: 2012PLoSO...730809I. 
  12. ↑ Takahashi, Hazuki (2012). "5' end-centered expression profiling using cap-analysis gene expression and next-generation sequencing.". Nat Protoc 7 (3): 542–61. doi:10.1038/nprot.2012.005. PMID 22362160. 
  13. ↑ Batut, Philippe (2013). "High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression.". Genome Res. 23 (1): 169–80. doi:10.1101/gr.139618.112. PMID 22936248. 
  14. ↑ Murata, Mitsuyoshi (2014). "Detecting Expressed Genes Using CAGE". Transcription Factor Regulatory Networks. Methods in Molecular Biology. 1164. pp. 67–85. doi:10.1007/978-1-4939-0805-9_7. ISBN 978-1-4939-0804-2. 
  15. ↑ Poulain, Stéphane (2017). "NanoCAGE: A Method for the Analysis of Coding and Noncoding 5′-Capped Transcriptomes". Promoter Associated RNA. Methods in Molecular Biology. 1543. pp. 57–109. doi:10.1007/978-1-4939-6716-2_4. ISBN 978-1-4939-6714-8. 
  16. ↑ Cvetesic, Nevena (2018). "SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA". Genome Research 28 (12): 1943–1956. doi:10.1101/gr.235937.118. PMID 30404778. 
  17. ↑ Takahashi, Hazuki (2021). "Low Quantity Single Strand CAGE (LQ-ssCAGE) Maps Regulatory Enhancers and Promoters". Enhancers and Promoters. Methods Mol Biol.. 2351. pp. 67–90. doi:10.1007/978-1-0716-1597-3_4. ISBN 978-1-0716-1596-6. 

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