Allele Specific Expression (ASE) analysis between Bos Taurus and Bos Indicus cows using RNA-Seq data at SNP level and gene level.

In recent years, large-scale international studies have provide comprehensive catalogues of genomic alterations in cancers including Esophageal Squamous Cell Cancer(ESCC). They revealed that some gene associated with cell cycle/apoptosis pathway, NOTCH pathway, WNT pathway, such as TP53 and NOTCH1, harbored genetic abnormalities frequently. As the next step clinical sequencing studies are starting to evaluate efficacy of using targeted agents to patients with specific molecular aberrations. We performed exome sequencing and RNA sequencing for 25 Japanese patients with esophageal squamous cell carcinoma (ESCC) to provide a comprehensive catalogue of genomic abnormalities in ESCC and found TP53 and ZNF750 significantly mutated genes. Additionally, we performed allele specific expression analysis of TP53, integrating mRNA sequencing data into the information of genomic abnormality. This analysis revealed that levels of expression changes depending on mutation types and nearly mono-allelic expression of TP53 was a common signature of ESCC patients with somatic mutations. And pattern of mono-allelic expression was dependent on mutation types. We expanded this analysis to all genes with somatic SNV mutations and revealed that mutant allele specific expression was observed in other genes including ZNF750, and many of them were belonged to cancer pathway in KEGG database. About TP53, our investigation might provide better understanding of the involvement of somatic mutations. And fluctuations in transcriptional regulation of TP53 could be predicted based on type of somatic mutation. In addition to this, analysis of allele specific expression suggested that not only somatic mutation of DNA, but also mutant allele expression should be considered to understand cancer genetic pathophysiology better and build more effective therapeutic strategies. Citation Format: Masahiko Takahashi, Hirofumi Nakaoka, Yasunori Akutsu, Naoyuki Hanari, Kentaro Murakami, Masayuki Kano, Yasunori Matsumoto, Ryota Otsuka, Nobufumi Sekino, Masaya Yokoyama, Itsuro Inoue, Hisahiro Matsubara. Analysis of allele specific expression in esophageal squamous cell carcinoma with combination of exome sequencing and mRNA Sequencing [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B07.

BackgroundGenetic analysis of gene expression level is a promising approach for characterizing candidate genes that are involved in complex economic traits such as meat quality. In the present study, we conducted expression quantitative trait loci (eQTL) and allele-specific expression (ASE) analyses based on RNA-sequencing (RNAseq) data from the longissimus muscle of 189 Duroc × Luchuan crossed pigs in order to identify some candidate genes for meat quality traits.ResultsUsing a genome-wide association study based on a mixed linear model, we identified 7192 cis-eQTL corresponding to 2098 cis-genes (p ≤ 1.33e-3, FDR ≤ 0.05) and 6400 trans-eQTL corresponding to 863 trans-genes (p ≤ 1.13e-6, FDR ≤ 0.05). ASE analysis using RNAseq SNPs identified 9815 significant ASE-SNPs in 2253 unique genes. Integrative analysis between the cis-eQTL and ASE target genes identified 540 common genes, including 33 genes with expression levels that were correlated with at least one meat quality trait. Among these 540 common genes, 63 have been reported previously as candidate genes for meat quality traits, such as PHKG1 (q-value = 1.67e-6 for the leading SNP in the cis-eQTL analysis), NUDT7 (q-value = 5.67e-13), FADS2 (q-value = 8.44e-5), and DGAT2 (q-value = 1.24e-3).ConclusionsThe present study confirmed several previously published candidate genes and identified some novel candidate genes for meat quality traits via eQTL and ASE analyses, which will be useful to prioritize candidate genes in further studies.

Single-cell RNA-seq (scRNA-seq) is emerging as a powerful tool for understanding gene function across diverse cells. Recently, this has included the use of allele-specific expression (ASE) analysis to better understand how variation in the human genome affects RNA expression at the single-cell level. We reasoned that because intronic reads are more prevalent in single-nucleus RNA-Seq (snRNA-Seq), and introns are under lower purifying selection and thus enriched for genetic variants, that snRNA-seq should facilitate single-cell analysis of ASE. Here we demonstrate how experimental and computational choices can improve the results of allelic imbalance analysis. We explore how experimental choices, such as RNA source, read length, sequencing depth, genotyping, etc., impact the power of ASE-based methods. We developed a new suite of computational tools to process and analyze scRNA-seq and snRNA-seq for ASE. As hypothesized, we extracted more ASE information from reads in intronic regions than those in exonic regions and show how read length can be set to increase power. Additionally, hybrid selection improved our power to detect allelic imbalance in genes of interest. We also explored methods to recover allele-specific isoform expression levels from both long- and short-read snRNA-seq. To further investigate ASE in the context of human disease, we applied our methods to a Parkinson’s disease cohort of 94 individuals and show that ASE analysis had more power than eQTL analysis to identify significant SNP/gene pairs in our direct comparison of the two methods. Overall, we provide an end-to-end experimental and computational approach for future studies.

Allele-specific expression (ASE) analysis improves the understanding of transcription’s cis-regulation. Herein, we used imputed SNPs along with RNA-Seq data from the Longissiumus thoracis muscle of 190 Nelore steers to identify functional cis-regulatory variants from ASE analysis. Using a Binomial Test, we identified 38,177 SNPs in ASE regions (ASE SNPs; FDR ≤0.05). We then searched for aseQTLs (SNPs potentially regulating the ASE) by comparing their heterozygosity to the measured allelic ratio under a Wilcoxon Rank Sum test. We identified 21,543 aseQTLs potentially regulating a total of 430 ASE SNPs (FDR ≤0.05). Based on a linear model, ASE SNPs and aseQTLs were associated with transcript abundance. We identified 3,333 SNPs acting as cis-eQTLs (FDR≤0.05). Results were integrated with previous ASE, functional regions, and meat quality-related differentially expressed genes data. This study described novel SNPs potentially regulating the transcription of genes that may affect beef traits.

Cancer has long thought to be a disease which develops from de novo cancer driver mutations in oncogenes and tumor suppressor genes (TSGs). The purpose of our study is to examine how TSGs contribute to tumorigenesis. Accordingly, we studied loss-of-function (LoF) mutations in TSGs within 2504 individuals from 1000 Genomes Project (1KGP) and 233 patients across four different cancer types from The Cancer Genome Atlas (TCGA). We found a large fraction of 1KGP individuals were carriers of heterozygous LoF mutations in TSGs. However, compound heterozygosity of LoF mutations in at least one TSG was only found in 20% of our TCGA patients. Further, analysis of allele specific expression (ASE) in these tumors identified several TSGs where the mutant allele is overexpressed relative to the reference allele. This evidence of ASE suggests TSGs have the potential to drive tumorigenesis in the heterozygous condition, if the reference allele is sufficiently repressed. Citation Format: Evan Clayton. Tumor suppressor genes may promote tumorigenesis through allele specific expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3388.

The aim of this study was to compare the length and intensity of oestrus in nonlactating Bos taurus (Holstein; n = 11) v. nonlactating Bos indicus (Nelore; n = 13) cows. The cows were kept in a single pen to allow interaction between them and were daily fed a maintenance diet according to the NRC (2000), containing 42.8% sugarcane bagasse, 45.7% corn, 3.2% soybean meal, 1.2% urea, 5.7% molasses, and 1.4% mineral salt, totaling 71% dry matter (DM) in the diet. The intake of DM per kg of body weight (BW) was 1.45%. Body condition score and BW were 3.5 ± 0.1 and 3.0 ± 0.2 (range 1 to 5), and 549.5 ± 14.3 and 625.5 ± 20.5 kg for Nelore and Holstein cows, respectively. All females had the emergence of follicular wave synchronized with an intravaginal progesterone device (Sincrogest, Ourofino Agronegócio, São Paulo, Brazil), 2 mg of oestradiol benzoate (Sincrodiol, Ourofino; IM) and 0.150 mg PGF2α (Sincrocio, Ourofino; IM). Ten days after initiation of the protocol, the intravaginal device was removed and another treatment with 0.150 mg PGF2α was given. Thereafter, cows were evaluated for oestrus by continuous visual observation 24 h a day for 5 days. The number of the cow that was mounting and the number of the cow that was standing was recorded for each event, as well as the time of the event. To confirm ovulation, ultrasound examination was performed between 5 and 7 days after oestrus detection. Data were analysed by Student’s t-test and Fisher’s exact test or Chi-square and data are presented as mean ± SE or percentage. Ten of 13 (76.9%) and 11 of 11 (100%) Nelore and Holstein cows, respectively, were detected in standing oestrus and ovulated (P > 0.10). On average, Holstein cows tended to start oestrus earlier than Nelore cows after device removal (40.4 ± 2.9 v. 47.7 ± 2.8 h; P = 0.09). There was no difference in intensity and duration of oestrus between Bos taurus and Bos indicus cows. Oestrus length was 14.7 ± 1.0 h (range 8.3 to 19.0 h) in Holstein cows and 12.4 ± 0.8 h (range 8.6 to 17.1 h) in Nelore cows (P > 0.10). Moreover, the average number of times that cows accepted mounts was 32.2 ± 6.1 and 36.3 ± 5.3 for Holstein and Nelore cows, respectively (P > 0.10). Only 9.1% of the mounts accepted by Nelore cows were performed by Holstein cows and only 3.8% of the mounts accepted by Holstein cows were done by Nelore cows (P < 0.05). We concluded that Bos taurus and Bos indicus cows managed under similar environment and nutritional conditions exhibit oestrus with the same intensity and duration. However, there was very little interaction during oestrus between Nelore and Holstein cows, showing a breed segregation pattern. This work was financially supported by CNPq, FAPESP, and Ourofino Agronegócio of Brazil.

Allele-specific expression (ASE) analysis detects the relative abundance of alleles at heterozygous loci as a proxy for cis-regulatory variation, which affects the personal transcriptome and proteome. This study describes the development and application of an ASE analysis pipeline on a unique cohort of 87 well phenotyped and RNA sequenced patients from the Maastricht Cardiomyopathy Registry with dilated cardiomyopathy (DCM), a complex genetic disorder with a remaining gap in explained heritability. Regulatory processes for which ASE is a proxy might explain this gap. We found an overrepresentation of known DCM-associated genes among the significant results across the cohort. In addition, we were able to find genes of interest that have not been associated with DCM through conventional methods such as genome-wide association or differential gene expression studies. The pipeline offers RNA sequencing data processing, individual and population level ASE analyses as well as group comparisons and several intuitive visualizations such as Manhattan plots and protein–protein interaction networks. With this pipeline, we found evidence supporting the case that cis-regulatory variation contributes to the phenotypic heterogeneity of DCM. Additionally, our results highlight that ASE analysis offers an additional layer to conventional genomic and transcriptomic analyses for candidate gene identification and biological insight.

Background: Genome-wide association study (GWAS) have identified over 45 susceptibility loci for lung cancer; many studies including our own group, have focused on low-frequency and rare coding variants using fine mapping and exome sequencing. This strategy, however, has met with limited success as about 90% of GWAS hits are noncoding and act primarily through altering transcriptional regulation in an allele-specific manner. The RNA-Seq based allele-specific expression (ASE) analysis affords an innovative approach to study preferential expression of an allele in direct relationship to its genotype, providing information on cis-regulatory effects for the expression of putative genes. However currently, there are no lung cancer studies that have rigorously evaluated the ASE variation in lung tumor and adjacent tissues. Methods: Leveraging The Cancer Genome Atlas (TCGA) resource, we performed transcriptomic-wide ASE analysis using existing RNA-Seq datasets of paired tumor and adjacent tissues from 54 lung adenocarcinoma patients. We first quantified the RNA read counts of Referent and Alternate alleles of heterozygous variants, then evaluated the allelic imbalance on a per-sample basis using Beta-binomial test, and explored the differential ASE between tumor and adjacent tissues using paired Wilcoxon test. Functional regulatory consequences were generated from Ensembl Variant Effect Predictor. Results: We identified total 208 significant ASEs, including 35 tissue-specific (only in tumor or only in adjacent), 28 sharing, and 145 differential variants. Of the 208 candidates, 41 were from the human leukocyte antigen (HLA) locus (primary DQA2, DQB1, DRB1, H and J), 26 were from the immunoglobulin (IG) superfamily (primary IGH, IGL, IGK and F11R). About 80% candidates were noncoding (mostly in 5’ and 3’ untranslated regions) and with regulatory features (21 promoter, seven enhancer, seven open chromatin region, two induce nonsense-mediated mRNA decay, one CTCF-binding site, and one transcription factor binding site). Other top genes included MDM2, APOL1, and CTSB. Pathway analyses revealed 27 genes involved in immune response pathway, and 12 genes involved in HLA antigen processing and presentation pathway. Conclusion: This study is the first transcriptomics ASE analysis in lung adenocarcinoma. The key somatic cis-regulatory ASE variants identified from this study, especially immunogenic allelic variations from HLA and IG genes, could be used for identifying high-risk individuals for targeted lung cancer checkpoint blockade and related immunotherapies. Citation Format: Yanhong Liu, Spiridon Tsavachidis, Farrah Kheradmand, Margaret R. Spitz, Chris Amos. Transcriptome analysis links immune genes allelic expression imbalances to lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1584.

Combining allele-specific expression (ASE) analysis with single-cell RNA-seq can elucidate how genomic variation affects RNA expression at the single-cell level. We explore how experimental and computational choices impact the power of ASE-based methods and develop a suite of single-cell ASE computational tools. With single-nucleus RNA-Seq, we extract more ASE information from reads in intronic than exonic regions. We show how read length can increase power and that hybrid selection improves power to detect ASE in targeted genes. We apply our methods to a Parkinson's disease cohort and show that ASE analysis has more power than eQTL analysis.

Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.

BackgroundThe simplest definition of cis-eQTLs versus trans, refers to genetic variants that affect expression in an allele specific manner, with implications on underlying mechanism. Yet, due to technical limitations of expression microarrays, the vast majority of eQTL studies performed in the last decade used a genomic distance based definition as a surrogate for cis, therefore exploring local rather than cis-eQTLs.ResultsIn this study we use RNAseq to explore allele specific expression (ASE) in adipose tissue of male and female F1 mice, produced from reciprocal crosses of C57BL/6J and DBA/2J strains. Comparison of the identified cis-eQTLs, to local-eQTLs, that were obtained from adipose tissue expression in two previous population based studies in our laboratory, yields poor overlap between the two mapping approaches, while both local-eQTL studies show highly concordant results. Specifically, local-eQTL studies show ~60% overlap between themselves, while only 15-20% of local-eQTLs are identified as cis by ASE, and less than 50% of ASE genes are recovered in local-eQTL studies. Utilizing recently published ENCODE data, we also find that ASE genes show significant bias for SNPs prevalence in DNase I hypersensitive sites that is ASE direction specific.ConclusionsWe suggest a new approach to analysis of allele specific expression that is more sensitive and accurate than the commonly used fisher or chi-square statistics. Our analysis indicates that technical differences between the cis and local-eQTL approaches, such as differences in genomic background or sex specificity, account for relatively small fraction of the discrepancy. Therefore, we suggest that the differences between two eQTL mapping approaches may facilitate sorting of SNP-eQTL interactions into true cis and trans, and that a considerable portion of local-eQTL may actually represent trans interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-471) contains supplementary material, which is available to authorized users.

Genes with parent-of-origin specific expression are not as numerous as previously reported and validated parentally-biased expressed genes tend to be peripheral to known imprinted domains indicating shared regional control.

Genes with parent-of-origin specific expression are not as numerous as previously reported and validated parentally-biased expressed genes tend to be peripheral to known imprinted domains indicating shared regional control.

BackgroundAllele specific expression (ASE) has become an important phenotype, being utilized for the detection of cis-regulatory variation, nonsense mediated decay and imprinting in the personal genome, and has been used to both identify disease loci and consider the penetrance of damaging alleles. The detection of ASE using high throughput technologies relies on aligning short-read sequencing data, a process that has inherent biases, and there is still a need to develop fast and accurate methods to detect ASE given the unprecedented growth of sequencing information in big data projects.ResultsHere, we present a new approach to normalize RNA sequencing data in order to call ASE events with high precision in a short time-frame. Using simulated datasets we find that our approach dramatically improves reference allele quantification at heterozygous sites versus default mapping methods and also performs well compared to existing techniques for ASE detection, such as filtering methods and mapping to parental genomes, without the need for complex and time consuming manipulation. Finally, by sequencing the exomes and transcriptomes of 96 well-phenotyped individuals of the CARTaGENE cohort, we characterise the levels of ASE across individuals and find a significant association between the proportion of sites undergoing ASE within the genome and smoking.ConclusionsThe correct treatment and analysis of RNA sequencing data is vital to control for mapping biases and detect genuine ASE signals. By normalising RNA sequencing information after mapping, we show that this approach can be used to identify biologically relevant signals in personal genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1238-8) contains supplementary material, which is available to authorized users.

First service fertility is an important factor affecting the calving-conception interval and the number of services per conception. In the present study, first service fertility of 618 multiparous dual purpose cows was analyzed according to breed predominance (Bos indicus and Bos taurus) and service season: dry (December-March); sub-humid (April-August); and humid season (September-November). All cows were located in a sub-humid tropical forest area, characterized by 1950 mm/year of rainfall and a mean daily temperature of 28.3�C. Cows were milked and suckled twice a day. Estrus was detected twice a day with a minimum observation period of 30 min. All cows were serviced according to the international rule AM-PM with semen of one of two Brahman bulls (A and B). All cows received water and mineral salt ad libitum during the entire year. Additionally, during the dry season cows received an energy supplementation. Data were analyzed using the chi-square procedure of SAS (SAS/STAT User's Guide, 8.2 ed. Cary, NC: SAS Institute, Inc., 2001). No bull effects were observed on first service fertility (bull A: 37.70%, 105/305; and bull B: 40.26%, 126/313; P > 0.05). Breed predominance significantly affected the first service fertility, being higher in Bos indicus cows (47.48%) than in Bos taurus cows (32.78%; P < 0.05). The higher first service fertility of Bos indicus cows seen during the year was probably because Bos indicus cattle are more thermotolerant than Bos taurus cattle (Hansen 2004 Anim. Reprod. Sci. 82-83, 349-360). Moreover, season of service did not affect the first service fertility of Bos indicus cows (dry: 48.95%, 70/143; sub-humid: 50%, 33/66; and humid: 41.30%, 19/46; P > 0.05). While in Bos taurus cows, first service fertility was higher in cows serviced during the dry season (43.24%) in comparison with those serviced during the sub-humid (28.26%; P < 0.05) or humid season (23.58%; P < 0.05). No differences were observed between Bos taurus cows serviced in the sub-humid and the humid season (P > 0.05). First service fertility did not differ between Bos indicus and Bos taurus cows serviced during the dry season (P > 0.05), which is likely due to the lower relative humidity and extra energy supplementation. This may improve oocyte quality. In conclusion, fertility of Bos taurus cattle is more sensitive than that of Bos indicus cattle under tropical conditions; therefore, the implementation of a reproductive seasonality scheme is recommended to increase the reproductive efficiency of Bos taurus cattle.