A droplet digital PCR detection method for rare L1 insertions in tumors

White, Travis B.; McCoy, Adam M.; Streva, Vincent A.; Fenrich, Joshua; Deininger, Prescott L.

doi:10.1186/s13100-014-0030-4

Cited by 19 publications

(15 citation statements)

References 34 publications

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“…2C, and Table S5). Digital PCR overcame the limitations of standard PCR assays to accurately detect rare nucleic acid sequences 7,21 . The digital PCR assay involved partitioning samples into a large number of small droplets prior to amplification.…”

Section: Somatic L1 Retrotransposon Insertions Occur In Progenitors Cmentioning

confidence: 99%

L1-associated genomic regions are deleted in somatic cells of the healthy human brain

et al. 2016

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The healthy human brain is a mosaic of varied genomes. L1 retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that Somatic L1-Associated Variants (SLAVs) are actually composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs are, in fact, somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition- independent rearrangements within inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2/PSD93, and affect between 44–63% of cells of the cells in the healthy brain.

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Section: Somatic L1 Retrotransposon Insertions Occur In Progenitors Cmentioning

confidence: 99%

L1-associated genomic regions are deleted in somatic cells of the healthy human brain

et al. 2016

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“…Recent technological advances, including next-generation sequencing, now allow for comprehensive analysis of somatic retrotranspositions [70,71]. A number of tools have been developed in the last several years to analyze L1 insertions in both normal tissues and tumors, including ME-Scan [72], droplet digital PCR [73], and Transposeq [74]. Similarly, tools such as RepEnrich have been developed to study genome-wide transcriptional regulation of TEs [75].…”

Section: Introductionmentioning

confidence: 99%

Response of transposable elements to environmental stressors

Miousse

Chalbot

Lumen

et al. 2015

Mutation Research/Reviews in Mutation Research

125

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Transposable elements (TEs) comprise a group of repetitive sequences that bring positive, negative, as well as neutral effects to the host organism. Earlier considered as “junk DNA,” TEs are now well-accepted driving forces of evolution and critical regulators the of expression of genetic information. Their activity is regulated by epigenetic mechanisms, including methylation of DNA and histone modifications. The loss of epigenetic control over TEs, exhibited as loss of DNA methylation and decondensation of the chromatin structure, may result in TEs reactivation, initiation of their insertional mutagenesis (retrotransposition) and has been reported in numerous human diseases, including cancer. Accumulating evidence suggests that these alterations are not the simple consequences of the disease, but often may drive the pathogenesis, as they can be detected early during disease development. Knowledge derived from the in vitro, in vivo, and epidemiological studies, clearly demonstrates that exposure to ubiquitous environmental stressors, many of which are carcinogens or suspected carcinogens, are capable of causing alterations in methylation and expression of TEs and initiate retrotransposition events. Evidence summarized in this review suggests that TEs are the sensitive endpoints for detection of effects caused by such environmental stressors, as ionizing radiation (terrestrial, space, and UV-radiation), air pollution (including particulate matter [PM]-derived and gaseous), persistent organic pollutants, and metals. Furthermore, the significance of these effects is characterized by their early appearance, persistence and presence in both, target organs and peripheral blood. Altogether, these findings suggest that TEs may potentially be introduced into safety and risk assessment and serve as biomarkers of exposure to environmental stressors. Furthermore, TEs also show significant potential to become invaluable surrogate biomarkers in clinic and possible targets for therapeutic modalities for disease treatment and prevention.

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“…The addition of purified host factors to these reactions would allow investigation of direct effects on ORF2p reverse transcriptase activity while avoiding some of the issues described herein. Next-generation sequencing methods [85, 88] including retrotransposon capture sequencing (RC-seq) [89, 90], as well as novel approaches for validation such as droplet digital PCR [91], offer the possibility of examining endogenous L1 elements in their native chromatin environment. These technical advances should facilitate investigation of the host factors that delimit L1 tissue specificity and various aspects of retrotransposition.…”

Section: Discussionmentioning

confidence: 99%

Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

Cook

Tabor

2016

Mobile DNA

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BackgroundThe Long INterspersed Element-1 (L1, LINE-1) is the only autonomous mobile DNA element in humans and has generated as much as half of the genome. Due to increasing clinical interest in the roles of L1 in cancer, embryogenesis and neuronal development, it has become a priority to understand L1-host interactions and identify host factors required for its activity. Apropos to this, we recently reported that L1 retrotransposition in HeLa cells requires phosphorylation of the L1 protein ORF1p at motifs targeted by host cell proline-directed protein kinases (PDPKs), which include the family of mitogen-activated protein kinases (MAPKs). Using two engineered L1 reporter assays, we continued our investigation into the roles of MAPKs in L1 activity.ResultsWe found that the MAPK p38δ phosphorylated ORF1p on three of its four PDPK motifs required for L1 activity. In addition, we found that a constitutively active p38δ mutant appeared to promote L1 retrotransposition in HeLa cells. However, despite the consistency of these findings with our earlier work, we identified some technical concerns regarding the experimental methodology. Specifically, we found that exogenous expression of p38δ appeared to affect at least one heterologous promoter in an engineered L1 reporter, as well as generate opposing effects on two different reporters. We also show that two commercially available non-targeting control (NTC) siRNAs elicit drastically different effects on the apparent retrotransposition reported by both L1 assays, which raises concerns about the use of NTCs as normalizing controls.ConclusionsEngineered L1 reporter assays have been invaluable for determining the functions and critical residues of L1 open reading frames, as well as elucidating many aspects of L1 replication. However, our results suggest that caution is required when interpreting data obtained from L1 reporters used in conjunction with exogenous gene expression or siRNA.

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A droplet digital PCR detection method for rare L1 insertions in tumors

Cited by 19 publications

References 34 publications

L1-associated genomic regions are deleted in somatic cells of the healthy human brain

L1-associated genomic regions are deleted in somatic cells of the healthy human brain

Response of transposable elements to environmental stressors

Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition

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