Abstract
BackgroundThe active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. Through its mobility via a process called target primed reverse transcription (TPRT), L1 mobilization has resulted in over 100 de novo cases of human disease and has recently been associated with various cancer types. Large advances in high-throughput sequencing (HTS) technology have allowed for an increased understanding of the role of L1 in human cancer; however, researchers are still limited by the ability to validate potentially rare L1 insertion events detected by HTS that may occur in only a small fraction of tumor cells. Additionally, HTS detection of rare events varies greatly as a function of read depth, and new tools for de novo element discovery are needed to fill in gaps created by HTS.ResultsWe have employed droplet digital PCR (ddPCR) to detect rare L1 loci in mosaic human genomes. Our assay allows for the detection of L1 insertions as rare as one cell in every 10,000.ConclusionsddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-014-0030-4) contains supplementary material, which is available to authorized users.
Highlights
The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome
Because tumors are often heterogeneous in genomic content, discovery and validation of de novo L1 insertion events detected by high-throughput sequencing (HTS) in tumors can be problematic [13]
Through separation of DNA templates for PCR in up to 20,000 droplets and measurement of fluorescence for each droplet at the terminal plateau phase of PCR, droplet digital PCR (ddPCR)-based L1 detection is capable of a high degree of discrimination that is not possible with standard TaqManTM assays [14]
Summary
The active human mobile element, long interspersed element 1 (L1) currently populates human genomes in excess of 500,000 copies per haploid genome. The human retrotransposon, long interspersed element 1 (L1) exists in over half a million copies per genome and constitutes 17% of genomic content [1]. The majority of these copies are nonfunctional relics that litter the genome; on average, approximately 100 L1 elements remain active in any given individual [1,2]. Because tumors are often heterogeneous in genomic content, discovery and validation of de novo L1 insertion events detected by HTS in tumors can be problematic [13].
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