Top ASE genes from the liver tissue sample.

<p>^a Imprinting status sourced from <a href="http://www.geneimprint.org/" target="_blank">http://www.geneimprint.org/</a> accessed December 4, 2013.</p><p>^b eQTL, transcript-QTL and exon-QTL data sourced from <a href="http://eqtl.uchicago.edu/cgi-bin/gbrowse/eqtl/" target="_blank">http://eqtl.uchicago.edu/cgi-bin/gbrowse/eqtl/</a>, accessed January 29, 2014.</p><p>Top ASE genes from the liver tissue sample.</p

SNPs proximal to SNVs are more likely to be ASE.

<p>(A) SNPs binned by genomic distance to nearest heterozygous indel. (B) SNPs binned by nearest heterozygous un-phased <i>de novo</i> called SNP. (C) SNPs binned by the number of proximal heterozygous SNPs. (D) SNPs binned by genomic alignability. The closer a SNP is to a heterozygous SNV, the more likely that SNP is to be classified as ASE. For indels, this effect is stronger than SNPs, which are only slightly affected, however as the number of SNPs within a read length (100bp) increases, this effect is increased. In all plots, the dashed line represents the average ASE proportion for all SNPs. Numbers at the top of the bars indicate the number of SNPs in each bin (blue not ASE, red ASE).</p

Proportion of SNPs annotated within transcript features categorised as ASE.

<p>Dashed black line represents the average proportion of all ASE SNPs. SNPs falling within intergenic regions (p < 0.005) are more likely to be classified ASE than SNPs falling in other regions. This observation is most likely due to the high amount of imprinted SNPs in the brain sample that do not have genic annotations.</p

Reference fraction correlations between replicates using diploid alignment.

<p>Each point is a SNP, coloured according to its ASE classification in both replicates. Bar plots indicate the proportions of all SNPs in the plot (left), and ASE SNPs (right). Calculating ASE with low read counts can result in sampling being a confounder.</p

Putative allele-specific isoform expression.

<p>Red circles are exonic SNPs, blue circles intronic SNPs, gray circles are filtered SNPs (either poor alignability or SNV proximity). SNP colour is proportional to probability of ASE (darker is more significant). Error bars represent the 95% binomial confidence interval (Pearson-Klopper). The gene model at the top shows the exonic (red) and intronic (blue) SNPs, the black sticks above these represent the read depth (min height represents 10 reads, max height 200 or more reads). Isoform models below are ordered by expression (top highest), and coloured by expression (darker is higher expressed). Only exonic SNPs are shown on transcripts, and only transcripts with at least one testable exonic SNP are drawn. Exon lengths are drawn at log2, and intron lengths at log10. (A) At the <i>CFHR3</i> locus in the liver sample, two SNPs flanking either end of the gene show high allele A expression, three of which are specific to isoform <i>CFHR3-001</i>. Five SNPs show lower allele A expression in the fifth exon, and are specific to isoform <i>CFHR3-005</i>. The intronic SNPs show skewed expression towards allele B, and suggest allele-specific transcription. Taken together, these data suggest that both isoforms <i>CFHR3-001</i> and <i>CFHR3-003</i> are ASE, even though the gene as a whole does not show evidence of ASE. (B) In the liver sample, the ubiquitously expressed gene <i>LGALS8</i> shows isoform specific ASE from transcript <i>LGALS8</i>-009 (towards allele A), whereas isoform <i>LGALS8</i>-001 shows balanced expression. (C) <i>SLCO1A2</i> is a brain specific anion-transporting polypeptide, and in our brain sample it expresses two isoforms testable for ASE. Both isoforms overlap specific SNPs that show imbalanced allelic expression in opposite directions in their respective 3’UTRs. Isoform <i>SLCO1A2</i>-202 appears imbalanced towards haplotype B, and isoform <i>SLCO1A2</i>-004 towards haplotype A.</p

Recommended workflow for ASE quantification using matched genotype and RNA-seq data.

<p>Robust quantification of ASE requires detailed genotype information and matched high quality RNA-seq data. Phasing and imputing SNPs provides haplotype information, used to determine whether intragenic SNPs with imbalanced expression show concordant direction of imbalance, as well as build personalised reference genomes for alignment. Personalised alignment increases both sensitivity (data yield) and specificity of ASE quantification. WGS data can be used to identify indels and other proximal SNVs not included in the imputation reference data set that may confound alignment and potentially produce false positive ASE calls. SNPs proximal to such factors can be removed from further analysis. Aggregation of SNPs by gene and isoform boundaries reduces potential sampling issues for lowly expressed regions, and assists in identifying ASE regions (genes and isoforms).</p

Schematic representation of three novel ASE genes in liver tissue.

<p>Red circles are exonic SNPs, blue circles intronic SNPs. SNP colour is proportional to probability of ASE (darker is more significant). Error bars represent the 95% binomial confidence interval (Pearson-Klopper). The gene model at the top shows the exonic (red) and intronic (blue) SNPs, the black sticks above these represent the read depth (minimum height represents 10 reads, maximum height 200 or more reads). Isoform models below are ordered by expression (top highest), and coloured by expression (darker is higher expressed). Only exonic SNPs are shown on transcripts, and only transcripts with at least one testable exonic SNP are drawn. Exon lengths are drawn at log2, and intron lengths at log10. Desmoplakin (<i>DSP</i>) is shown in panel (A), WD Repeat Domain 72 (<i>WDR72</i>) in panel (B) and Ubiquitin D (<i>UBD</i>) in panel (C). All three genes contain multiple imbalanced SNPs concordant with haplotype phase, indicating strong ASE. Multiple intronic SNPs were testable in <i>WDR72</i> and also show strong imbalance, suggesting that this gene was likely transcribed from a single allele. As no intronic SNPs were testable in either <i>DSP</i> or <i>UBD</i>, we cannot determine in these cases whether these genes were subject to allele-specific transcription or allele-specific post-transcriptional regulation.</p

Highest ranked ASE genes from (A) brain and (B) liver.

<p>Each gene is shown as vertical bars of exonic SNPs testable within that gene. Red boxes are imbalanced towards haplotype A, and blue boxes imbalanced towards haplotype B, darker colours indicate more significant the level of imbalance. All data presented is the average of the two replicates. Genes are ordered left to right by the probability of gene imbalance. Stars below the bars indicate whether the gene is known imprinted (red), or whether the SNPs for that gene all show concordant direction of expression imbalance in each replicate (black). Each of the tissues has a cluster of genes with very significant SNPs mostly with concordant direction of ASE towards the left of the plots (strong genic ASE). It is clear, however, that some genes contain SNPs that vary in their direction and magnitude of imbalance.</p