RC-Seq
Retrotransposon Capture Sequencing
RC-seq is a high-throughput protocol to map and study retrotransposon insertions (Baillie et al., 2011). In this method, after gDNA is fractionated, retrotransposon binding sites on DNA hybridize to transposon binding sites on a microarray. Deep sequencing provides accurate information that can be aligned to a reference sequence to discover novel retrotransposition events. A single-cell version (scRC-seq) has also been described (Upton et al., 2015).
Advantages:
- Ability to clearly identify and detect novel retrotransposition events
- Can specifically study transposon binding sites of interest
- High-throughput protocol
- PCR validation rate estimated at 98.5%
Disadvantages:
- Different types of mobile element insertions (MEIs) require separate PCR experiments with different primers (Xing et al., 2013)
- Hybridization errors can lead to sequencing unwanted DNA fragments
- PCR biases can underrepresent GC-rich templates
- Similar transposition binding sites can lead to sequence ambiguity and detection for a transposition event
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Xing J., Witherspoon D. J. and Jorde L. B. Mobile element biology: new possibilities with high-throughput sequencing. Trends Genet. 2013;29:280-289
References:
Klawitter S., Fuchs N. V., Upton K. R., et al. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells. Nat Commun. 2016;7:10286
Upton K. R., Gerhardt D. J., Jesuadian J. S., et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell. 2015;161:228-239
Solyom S., Ewing A. D., Rahrmann E. P., et al. Extensive somatic L1 retrotransposition in colorectal tumors. Genome Res. 2012;22:2328-2338
Shukla R., Upton K. R., Munoz-Lopez M., et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell. 2013;153:101-111
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History: RC-Seq
Revision by sbrumpton on 2017-06-21 07:50:21 - Show/Hide
Retrotransposon Capture Sequencing
RC-seq is a high-throughput protocol to map and study retrotransposon insertions (Baillie et al., 2011). In this method, after gDNA is fractionated, retrotransposon binding sites on DNA hybridize to transposon binding sites on a microarray. Deep sequencing provides accurate information that can be aligned to a reference sequence to discover novel retrotransposition events. A single-cell version (scRC-seq) has also been described (Upton et al., 2015).
Advantages:- Ability to clearly identify and detect novel retrotransposition events
- Can specifically study transposon binding sites of interest
- High-throughput protocol
- PCR validation rate estimated at 98.5%
Disadvantages:- Different types of mobile element insertions (MEIs) require separate PCR experiments with different primers (Xing et al., 2013)
- Hybridization errors can lead to sequencing unwanted DNA fragments
- PCR biases can underrepresent GC-rich templates
- Similar transposition binding sites can lead to sequence ambiguity and detection for a transposition event
Reagents:Illumina Library prep and Array Kit SelectorReviews:Xing J., Witherspoon D. J. and Jorde L. B. Mobile element biology: new possibilities with high-throughput sequencing. Trends Genet. 2013;29:280-289References:Klawitter S., Fuchs N. V., Upton K. R., et al. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells. Nat Commun. 2016;7:10286Upton K. R., Gerhardt D. J., Jesuadian J. S., et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell. 2015;161:228-239Solyom S., Ewing A. D., Rahrmann E. P., et al. Extensive somatic L1 retrotransposition in colorectal tumors. Genome Res. 2012;22:2328-2338Shukla R., Upton K. R., Munoz-Lopez M., et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell. 2013;153:101-111