Abstract
Genome-wide association studies have discovered thousands of common alleles that associate with human phenotypes and disease. Many of these variants are in non-protein-coding (regulatory) regions and are believed to affect phenotypes by modifying gene expression. In any organism with a diploid genome, such as humans, measuring the expression of each allele of a gene provides a well-controlled way to identify allelic influences on that gene’s expression. Here, we describe a protocol for precisely measuring the allele-specific expression of individual genes. This method targets the nucleotide differences between the two alleles of a gene within an individual and measures the “allelic skew,” the extent to which one allele is expressed more than the other. We cover the design of effective assays, the optimization of reactions, and the interpretation of the resulting data.
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References
Hindorff LA, Sethupathy P, Junkins HA, Ramos EM, Mehta JP, Collins FS, Manolio TA (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 106:9362–9367. https://doi.org/10.1073/pnas.0903103106
Genomes Project C, Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA (2010) A map of human genome variation from population-scale sequencing. Nature 467:1061–1073. https://doi.org/10.1038/nature09534
Patwardhan RP, Hiatt JB, Witten DM, Kim MJ, Smith RP, May D, Lee C, Andrie JM, Lee SI, Cooper GM, Ahituv N, Pennacchio LA, Shendure J (2012) Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol 30:265–270. https://doi.org/10.1038/nbt.2136
Cowles CR, Hirschhorn JN, Altshuler D, Lander ES (2002) Detection of regulatory variation in mouse genes. Nat Genet 32:432–437. https://doi.org/10.1038/ng992
Chen R, Mias GI, Li-Pook-Than J, Jiang L, Lam HY, Chen R, Miriami E, Karczewski KJ, Hariharan M, Dewey FE, Cheng Y, Clark MJ, Im H, Habegger L, Balasubramanian S, O'Huallachain M, Dudley JT, Hillenmeyer S, Haraksingh R, Sharon D, Euskirchen G, Lacroute P, Bettinger K, Boyle AP, Kasowski M, Grubert F, Seki S, Garcia M, Whirl-Carrillo M, Gallardo M, Blasco MA, Greenberg PL, Snyder P, Klein TE, Altman RB, Butte AJ, Ashley EA, Gerstein M, Nadeau KC, Tang H, Snyder M (2012) Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell 148:1293–1307. https://doi.org/10.1016/j.cell.2012.02.009
Battle A, Mostafavi S, Zhu X, Potash JB, Weissman MM, McCormick C, Haudenschild CD, Beckman KB, Shi J, Mei R, Urban AE, Montgomery SB, Levinson DF, Koller D (2014) Characterizing the genetic basis of transcriptome diversity through RNA-sequencing of 922 individuals. Genome Res 24:14–24. https://doi.org/10.1101/gr.155192.113
Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74. https://doi.org/10.1093/nar/gkm306
Regan JF, Kamitaki N, Legler T, Cooper S, Klitgord N, Karlin-Neumann G, Wong C, Hodges S, Koehler R, Tzonev S, McCarroll SA (2015) A rapid molecular approach for chromosomal phasing. PLoS One 10:e0118270. https://doi.org/10.1371/journal.pone.0118270
Gray JM, Harmin DA, Boswell SA, Cloonan N, Mullen TE, Ling JJ, Miller N, Kuersten S, Ma YC, McCarroll SA, Grimmond SM, Springer M (2014) SnapShot-Seq: a method for extracting genome-wide, in vivo mRNA dynamics from a single total RNA sample. PLoS One 9:e89673. https://doi.org/10.1371/journal.pone.0089673
Acknowledgments
Our understanding of allelic skew has greatly benefited from the work of Tom Mullen and Jim Nemesh in our lab. This work was supported by a grant from the National Human Genome Research Institute (R01 HG006855, to SAM).
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Kamitaki, N., Usher, C.L., McCarroll, S.A. (2018). Using Droplet Digital PCR to Analyze Allele-Specific RNA Expression. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_23
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DOI: https://doi.org/10.1007/978-1-4939-7778-9_23
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