A droplet digital PCR detection method for rare L1 insertions in tumors - PubMed (original) (raw)

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

Travis B White et al. Mob DNA. 2014.

Abstract

Background: The 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.

Results: We 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.

Conclusions: ddPCR represents a robust method to be used alongside HTS techniques for detecting, validating and quantitating rare L1 insertion events in tumors and other tissues.

Keywords: L1; droplet digital PCR; retrotransposon; tumor.

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Figures

Figure 1

Figure 1

Schematic of L1Hs droplet digital PCR (ddPCR) assays. (A) A 5’-FAM labeled Taqman™ probe specific to the 5’ UTR of L1Hs is paired with an L1 5’ UTR-specific primer. The probe and primer anneal to complementary DNA strands. This primer/probe set can be used in conjunction with a unique genomic flanking primer to detect the 5’-insertion junction of specific full-length L1 elements in the human genome using ddPCR. A control assay using a primer and 5’-VIC labeled probe set specific to a housekeeping gene (RPP30) can be used in parallel to determine copy number. (B) A 5’-FAM labeled Taqman™ probe specific to the 3’ end of L1Hs is paired with an L1Hs-specific primer. The probe and primer anneal to complementary DNA strands. This primer/probe set can be used in conjunction with a unique genomic flanking primer to detect the 3’ insertion junction of specific L1Hs elements in the human genome using ddPCR.

Figure 2

Figure 2

Detection of chromosome 15 AC216176 L1Hs by the 5’ junction droplet digital PCR (ddPCR) assay. Each panel represents a single ddPCR experiment whereby a DNA sample (defined below) is segregated into individual droplets and assessed for the presence of the L1 locus (FAM) and RPP30 locus (VIC) using two different fluorophores in Taqman™ assays (see Figure 1). The FAM and VIC fluorescence for each droplet is plotted as a data point on each graph. FAM fluorescent signal (Channel 1) is plotted on the y-axis and VIC fluorescent signal (Channel 2) is plotted on the x-axis. The droplet threshold for each fluorophore used is indicated by the magenta lines, determining whether a droplet is considered positive or negative for either FAM or VIC fluorescence. The positive or negative fluorescence assessment for each quadrant is labeled accordingly for the plot describing the experiment with 100% GM01632 DNA. The blue dots represent individual droplets that contain at least one copy of the L1 locus tested but not the RPP30 locus (FAM positive, VIC negative), the green dots represent droplets that contain at least one copy of the RPP30 gene and not the L1 locus (VIC positive, FAM negative), and the orange dots represent droplets that contain at least one copy of both the RPP30 gene DNA and the L1 locus tested (positive for both FAM and VIC). We tested 160 ng of _BsaJI_-digested genomic DNA from GM01632 cells, which are homozygous for the polymorphic L1 element (100%), and tenfold dilutions of this same sample as a mixture with _BsaJI_-digested genomic DNA from GM01631 cells, which do not have this polymorphic L1 insertion (10%-0.01%), thus keeping the total input genomic DNA constant for each ddPCR. Additionally, as a negative control, 160 ng of _BsaJI_-digested genomic DNA from GM01631 cells was tested (0%).

Figure 3

Figure 3

Detection of chromosome 15 AC216176 L1Hs by the 3’ junction ddPCR assay. The L1Hs 3’ junction ddPCR assay uses a L1-specific primer, L1-specific 5’-FAM labeled Taqman™ probe, and a locus-specific primer near the Chromosome 15 AC216176 3’-insertion junction, as shown in Figure 1B. The FAM fluorescent signal (Ch 1) for each droplet is plotted on the y-axis for each of the ddPCR experiments, which are separated by a dotted yellow line, with input DNA indicated above each experiment. Each droplet is cumulatively counted as an ‘Event Number’ for the ddPCR experiments analyzed in tandem, and plotted along the x-axis. The positive droplet fluorescence threshold is indicated by the magenta line, which determines whether a droplet is considered positive or negative for FAM fluorescence. Thus, the blue dots represent individual droplets that contain at least one copy of the L1 locus tested. We tested 200 ng of _BamHI_-digested genomic DNA from HeLa cells, which contain the polymorphic L1 element, and tenfold dilutions of this same sample as a mixture with _BamHI_-digested genomic DNA from HEK293 cells, which do not have this polymorphic L1 insertion. Percentages given reflect the amount of input DNA with 100% corresponding to 200 ng of DNA. This assay robustly detects the 3’-insertion junction of the polymorphic full-length AC216176 L1Hs element when present in the genomic DNA from a cell line positive for that polymorphism (HeLa 100%), but not in a cell line negative for that polymorphism (HEK293 100%). L1-positive droplets are observed at dilutions as low as 0.01% of the DNA with this assay.

Figure 4

Figure 4

Concentration plot of chromosome 15 AC216176 L1Hs by the 3’ junction droplet digital PCR (ddPCR) assay. The input DNA concentrations in copies/μl (Ch1 Conc) for the ddPCR experiments described in Figure 3 were calculated by the QuantaSoft Analysis Software.

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References

    1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921. doi: 10.1038/35057062. - DOI - PubMed
    1. Brouha B, Schustak J, Badge RM, Lutz-Prigge S, Farley AH, Moran JV, Kazazian HH., Jr Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A. 2003;100:5280–5285. doi: 10.1073/pnas.0831042100. - DOI - PMC - PubMed
    1. Baillie JK, Barnett MW, Upton KR, Gerhardt DJ, Richmond TA, De Sapio F, Brennan PM, Rizzu P, Smith S, Fell M, Talbot RT, Gustincich S, Freeman TC, Mattick JS, Hume DA, Heutink P, Carninci P, Jeddeloh JA, Faulkner GJ. Somatic retrotransposition alters the genetic landscape of the human brain. Nature. 2011;479:534–537. doi: 10.1038/nature10531. - DOI - PMC - PubMed
    1. Ewing AD, Kazazian HH., Jr High-throughput sequencing reveals extensive variation in human-specific L1 content in individual human genomes. Genome Res. 2010;20:1262–1270. doi: 10.1101/gr.106419.110. - DOI - PMC - PubMed
    1. Ewing AD, Kazazian HH., Jr Whole-genome resequencing allows detection of many rare LINE-1 insertion alleles in humans. Genome Res. 2011;21:985–990. doi: 10.1101/gr.114777.110. - DOI - PMC - PubMed

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