Comprehensively Evaluating cis-Regulatory Variation in the Human Prostate Transcriptome by Using Gene-Level Allele-Specific Expression  Nicholas B. Larson, Shannon McDonnell, Amy J. French, Zach Fogarty, John Cheville, Sumit Middha, Shaun Riska, Saurabh Baheti, Asha A. Nair, Liang Wang, Daniel J. Schaid, Stephen N. Thibodeau  The American Journal of Human Genetics  Volume 96, Issue 6, Pages 869-882 (June 2015) DOI: 10.1016/j.ajhg.2015.04.015 Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 1 Using ASE to Detect cis-eQTLs (A) Example illustration of a cis-acting SNP that regulates gene expression. The width of the gray arrows indicates the magnitude of ASE. (B) Distributions of total expression for an example gene are based on the underlying cis-eQTL SNP genotype in the population. (C) Illustration of bioinformatics procedures utilized for quantifying TE and ASE measures with the use of RNA-seq and phased genotype data. Quantification of relative ASE was determined by the subset of total RNA-seq reads that overlapped eSNPs. The American Journal of Human Genetics 2015 96, 869-882DOI: (10.1016/j.ajhg.2015.04.015) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 2 Venn Diagrams of Tested cis-eQTL Associations and Gene-Level Significant Findings (A) Venn diagram depicting the overlap of unique SNP-gene pairs evaluated by each of the three cis-eQTL association methods (TE only, ASE only, and joint). (B) Venn diagram of genes with gene-wise significant eQTL associations per method. An overlapping result does not particularly indicate that the exact same SNP is the most significant eQTL for that gene across methods. The American Journal of Human Genetics 2015 96, 869-882DOI: (10.1016/j.ajhg.2015.04.015) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 3 Manhattan Plots of cis-eQTL Association p Values Manhattan plots depicting Bonferroni-adjusted p values of the strongest associated eQTLs for all (A) 16,570 genes under the TE method and (B) 13,998 genes under the joint testing method. Autosomes are ordered and alternately colored with dark and light gray. Genes in general are plotted as open circles, and solid green circles indicate genes identified as preferentially expressed in prostate tissue by TiGER. For display purposes, association results from prostate-specific genes with an adjusted p value < 1E−25 are labeled with a gene symbol. The American Journal of Human Genetics 2015 96, 869-882DOI: (10.1016/j.ajhg.2015.04.015) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 4 Example cis-eQTL Results for UPK3A Example cis-eQTL results for gene UPK3A, one of the top differentially expressed genes designated as preferentially expressed in prostate tissue. (A) –log10 p values of eQTL association results by base-pair position. Each SNP position corresponds to up to three test results, and the top associated SNP, rs2742629, is labeled and indicated by a filled symbol. (B) Boxplots of TE (indicated by raw total mapped reads) by the rs2742629 genotype. (C) This scatterplot of ASE coded by the phased rs2742629 genotype is based upon reads uniquely mappable to each phased haplotype. (D) An allelic-ratio plot of eSNPs (x axis) compares allelic expression ratios (y axis) for subjects homozygous for the rs2742629 genotype (cyan squares) and subjects heterozygous for the rs2742629 genotype (magenta circles). Each point represents a heterozygous eSNP (x axis) for a given sample, and the relative size of the plotting mark indicates the quantity of supporting ASE reads. Allelic bias is indicated by a deviation from 0.50 (horizontal dashed line). The American Journal of Human Genetics 2015 96, 869-882DOI: (10.1016/j.ajhg.2015.04.015) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 5 Properties of Significant cis-eQTL Associations (A) A density plot of the physical position of gene-wise significant results in relation to the TES and TSS of each gene is given for each association testing method. For display purposes, the distance between the TES and TSS is normalized to be 100 kb. (B) Scatterplot of absolute values of eQTL effect-size estimates by relative physical position for the gene-wise significant joint eQTL association results. (C) Annotation enrichment results for the PrEC ChromHMM chromatin states for all gene-level SNPs, as well as gene-region enrichment for the subset of these SNPs that occur within the transcribed region of the associated gene. The American Journal of Human Genetics 2015 96, 869-882DOI: (10.1016/j.ajhg.2015.04.015) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 6 Evidence of cis-Acting Co-regulation of Multiple Genes Illustration of cis-acting co-regulation of three separate genes by PRCA risk SNP rs684232. (A) Multi-gene regional association plot of eQTL association −log10 p values for SNPs within 200 kb of rs684232 for FAM57A (red square), GEMIN4 (green circle), and VPS53 (blue triangle); color intensity depicts LD with rs684232. Below is the UCSC Genome Browser display of genomic annotation for the accompanying region, including PrEC chromatin-state segmentation and DNase-seq peaks (the color scheme is based on UCSC ChromHMM display conventions), TEA enhancers, and AR binding sites. (B) Expression correlation matrices for the (left) TE (library-size adjusted) and (right) ASE allelic ratios for the three associated genes. Subjects were included for the latter if they had at least 20 ASE reads for each gene. The non-zero correlations for the allelic expression ratios indicate correlated gene expression specific to a given haplotype. (C) Boxplots of the allelic expression ratios for rs684232 (left) homozygotes and (right) heterozygotes. The ratio for heterozygotes is calculated as the rs684232 reference allele carrying haplotype expression divided by the sum of ASE reads per gene. The null expectation (0.50) is indicated by a dashed line. The heterozygote boxplots demonstrate the concurrent downregulatory effect attributed to the alternate allele across all three genes. The American Journal of Human Genetics 2015 96, 869-882DOI: (10.1016/j.ajhg.2015.04.015) Copyright © 2015 The American Society of Human Genetics Terms and Conditions