Expand your research: Next-Gen Sequencing, Genotyping, Gene Expression, and Epigenetics at the Center for Medical Genomics at IUSM

poster abstractThe Center for Medical Genomics (CMG) provides Indiana researchers with next-generation sequencing, SNP genotyping, gene expression and epigenetics. We provide expertise in experimental design, carry out the procedures, and assist with analyses and interpretation. These state-of-the-art technologies have enabled a large number of grants to be funded over the years, and have resulted in a very large number of publications. Our next-generation sequencing technology includes SOLiD5500xl, Ion Proton and Ion Torrent PGM (Personal Genome Machine). This set of instruments covers a wide range of nextgeneration sequencing capabilities from small bacterial genomes to the whole human genome, transcriptome (total RNA), small RNA, targeted DNA fragments, exome, ChIP-seq, and methylseq, with high sequencing accuracy. We have generated sequencing data for 74 projects over the past two-three years. Our SNP genotyping facility, using the Sequenom MassArray platform, specializes in targeted genotyping of 20-30 SNPs per assay and is an excellent choice for candidate gene studies and for following up results from GWAS and next-generation sequencing. It has been a central part of several large, multi-site collaborative genetic studies, including Genetics of Alcoholism (COGA), bipolar disorder, osteoporosis and hypertension, as well as many smaller projects; it is most efficient for sets of approximately 370 samples. We have produced more than 20 million targeted SNP genotypes to date. This platform is also capable of measuring allele-specific gene expression, and targeted quantitative DNA methylation for epigenetics study. For many projects, microarrays offer a good alternative to next-generation sequencing for measuring gene expression. We use Affymetrix GeneChip microarrays, capable of measuring expression of nearly all genes in humans (and all exons within them), rats, mice and most model organisms, and can measure expression of miRNAs. We can also use RNA extracted from FFPE samples. We have carried out more than 6,700 GeneChip hybridizations to date in support of many different projects. The CMG partners with the Center for Computational Biology and Bioinformatics for data analysis. We are recognized as a Core Facility of the Indiana CTSI and available to faculty not only from IU and IUPUI, but also from Purdue and Notre Dame Universities

High Glucose Alters Fetal Rat Islet Transcriptome and Induces Progeny Islet Dysfunction

Offspring of diabetic mothers are susceptible to developing type 2 diabetes due to pancreatic islet dysfunction. However, the initiating molecular pathways leading to offspring pancreatic islet dysfunction are unknown. We hypothesized that maternal hyperglycemia alters offspring pancreatic islet transcriptome and negatively impacts offspring islet function. We employed an infusion model capable of inducing localized hyperglycemia in fetal rats residing in the left uterine horn, thus avoiding other factors involved in programming offspring pancreatic islet health. While maintaining euglycemia in maternal dams and right uterine horn control fetuses, hyperglycemic fetuses in the left uterine horn had higher serum insulin and pancreatic beta cell area. Upon completing infusion from GD20 to 22, RNA sequencing was performed on GD22 islets to identify the hyperglycemia-induced altered gene expression. Ingenuity pathway analysis of the altered transcriptome found that diabetes mellitus and inflammation/cell death pathways were enriched. Interestingly, the downregulated genes modulate more diverse biological processes, which includes responses to stimuli and developmental processes. Next, we performed ex and in vivo studies to evaluate islet cell viability and insulin secretory function in weanling and adult offspring. Pancreatic islets of weanlings exposed to late gestation hyperglycemia had decreased cell viability in basal state and glucose-induced insulin secretion. Lastly, adult offspring exposed to in utero hyperglycemia also exhibited glucose intolerance and insulin secretory dysfunction. Together, our results demonstrate that late gestational hyperglycemia alters the fetal pancreatic islet transcriptome and increases offspring susceptibility to developing pancreatic islet dysfunction

RareVar: A Framework for Detecting Low-Frequency Single-Nucleotide Variants

Accurate identification of low-frequency somatic point mutations in tumor samples has important clinical utilities. Although high-throughput sequencing technology enables capturing such variants while sequencing primary tumor samples, our ability for accurate detection is compromised when the variant frequency is close to the sequencer error rate. Most current experimental and bioinformatic strategies target mutations with ≥5% allele frequency, which limits our ability to understand the cancer etiology and tumor evolution. We present an experimental and computational modeling framework, RareVar, to reliably identify low-frequency single-nucleotide variants from high-throughput sequencing data under standard experimental protocols. RareVar protocol includes a benchmark design by pooling DNAs from already sequenced individuals at various concentrations to target variants at desired frequencies, 0.5%-3% in our case. By applying a generalized, linear model-based, position-specific error model, followed by machine-learning-based variant calibration, our approach outperforms existing methods. Our method can be applied on most capture and sequencing platforms without modifying the experimental protocol

Ethanol Activates Immune Response In Lymphoblastoid Cells

The short term effects of alcohol on gene expression in brain tissue cannot directly be studied in humans. Because neuroimmune signaling is altered by alcohol, immune cells are a logical, accessible choice to study and might provide biomarkers. RNAseq was used to study the effects of 48 h exposure to ethanol on lymphoblastoid cell lines (LCLs) from 21 alcoholics and 21 controls. Ethanol exposure resulted in differential expression of 4,577 of the 12,526 genes detectably expressed in the LCLs (FDR ≤ 0.05); 55% of these showed increased expression. Cells from alcoholics and controls responded similarly. The genes whose expression changed fell into many pathways. NFκB, neuroinflammation, IL-6, and dendritic cell maturation pathways were activated, consistent with increased signaling by NFκB, TNF, TGFβ, IL1, IL4, IL18, TLR4, and LPS. Signaling by Interferons A and B decreased, which may be responsible for a slightly blunted immune response compared to 24 h ethanol treatment. EIF2, phospholipase C and VEGF signaling were decreased. Baseline gene expression patterns were similar in LCLs from alcoholics and controls. At relaxed stringency (p<0.05), 1164 genes differed, 340 of which were also affected by ethanol. There was a suggestion of compensation, with 77% showing opposing fold changes. Aldosterone signaling and phospholipase C signaling differed. The pattern of expression was consistent with increased signaling by several cytokines and TLR2 in alcoholics. The cholesterol biosynthesis pathway was lower in alcoholics, including a decrease in the rate-limiting enzyme HMGCR. LCLs show many effects of ethanol exposure, some of which might provide biomarkers for AUD and aid in interpreting the effects of genes identified by GWAS

Gene expression changes in the ventral hippocampus and medial prefrontal cortex of adolescent alcohol-preferring (P) rats following binge-like-alcohol drinking

Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including decreased hippocampal and prefrontal cortex volume and defects in memory. We used RNA sequencing to assess the effects of adolescent binge drinking on gene expression in these regions. Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-hour sessions/d during the dark/cycle, 5 days/week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 to 3 g/kg/session). Ethanol significantly altered the expression of 416 of 11,727 genes expressed in the ventral hippocampus. Genes and pathways involved in neurogenesis, long-term potentiation and axonal guidance were decreased, which could relate to the impaired memory function found in subjects with adolescent alcohol binge-like exposure. The decreased expression of myelin and cholesterol genes and apparent decrease in oligodendrocytes in P rats could result in decreased myelination. In the medial prefrontal cortex, 638 of 11,579 genes were altered; genes in cellular stress and inflammatory pathways were increased, as were genes involved in oxidative phosphorylation. Overall, the results of this study suggest that adolescent binge-like alcohol drinking may alter the development of the ventral hippocampus and medial prefrontal cortex and produce long-term consequences on learning and memory, and on control of impulsive behaviors

Gene expression changes in serotonin, GABA-A receptors, neuropeptides and ion channels in the dorsal raphe nucleus of adolescent alcohol-preferring (P) rats following binge-like alcohol drinking

Alcohol binge-drinking during adolescence is a serious public health concern with long-term consequences. We used RNA sequencing to assess the effects of excessive adolescent ethanol binge-drinking on gene expression in the dorsal raphe nucleus (DRN) of alcohol preferring (P) rats. Repeated binges across adolescence (three 1h sessions across the dark-cycle per day, 5 days per week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5-3 g/kg/session) significantly altered the expression of approximately one-third of the detected genes. Multiple neurotransmitter systems were altered, with the largest changes in the serotonin system (21 of 23 serotonin-related genes showed decreased expression) and GABA-A receptors (8 decreased and 2 increased). Multiple neuropeptide systems were also altered, with changes in the neuropeptide Y and corticotropin-releasing hormone systems similar to those associated with increased drinking and decreased resistance to stress. There was increased expression of 21 of 32 genes for potassium channels. Expression of downstream targets of CREB signaling was increased. There were also changes in expression of genes involved in inflammatory processes, axonal guidance, growth factors, transcription factors, and several intracellular signaling pathways. These widespread changes indicate that excessive binge drinking during adolescence alters the functioning of the DRN and likely its modulation of many regions of the central nervous system, including the mesocorticolimbic system

Effects of chronic intermittent ethanol exposure and withdrawal on neuroblastoma cell transcriptome

Cycles of heavy drinking and abstinence can lead to ethanol abuse disorder. We studied the effects of chronic intermittent ethanol exposure (CIE) over three weeks on neuroblastoma cells, using an ethanol concentration frequently attained in binge drinking (40 mM, 184 mg/dl). There were many changes in gene expression but most were small. CIE affected pathways instrumental in the development or plasticity of neurons, including axonal guidance, reelin signaling and synaptogenesis. Genes involved in dopamine and serotonin signaling were also affected. Changes in transporters and receptors could dampen both NMDA and norepinephrine transmissions. Decreased expression of the GABA transporter SLC6A11 could increase GABA transmission and has been associated with a switch from sweet drinking to ethanol consumption in rats. Ethanol increased stress responses such as unfolded protein response. TGF-β and NFκB signaling were increased. Most of the genes involved in cholesterol biosynthesis were decreased in expression. Withdrawal for 24 h after CIE caused most of the CIE-induced expression changes to move back toward unexposed levels

Gene expression changes in glutamate and GABA-A receptors, neuropeptides, ion channels and cholesterol synthesis in the periaqueductal gray following binge-like alcohol drinking by adolescent alcohol-preferring (P) rats

BACKGROUND: Binge drinking of alcohol during adolescence is a serious public health concern with long-term consequences, including increased pain, fear, and anxiety. The periaqueductal gray (PAG) is involved in processing pain, fear, and anxiety. The effects of adolescent binge drinking on gene expression in this region have yet to be studied. METHODS: Male adolescent alcohol-preferring (P) rats were exposed to repeated binge drinking (three 1-hour sessions/d during the dark/cycle, 5 days/wk for 3 weeks starting at 28 days of age; ethanol intakes of 2.5 to 3 g/kg/session). We used RNA sequencing to assess the effects of ethanol intake on gene expression. RESULTS: Ethanol significantly altered the expression of 1,670 of the 12,123 detected genes: 877 (53%) decreased. In the glutamate system, 23 genes were found to be altered, including reduction in 7 of 10 genes for metabotropic and NMDA receptors. Subunit changes in the NMDA receptor may make it less sensitive to ethanol. Changes in GABAA genes would most likely increase the ability of the PAG to produce tonic inhibition. Five serotonin receptor genes, 6 acetylcholine receptor genes, and 4 glycine receptor genes showed decreased expression in the alcohol-drinking rats. Opioid genes (e.g., Oprk1, Oprm1) and genes for neuropeptides linked to anxiety and panic behaviors (e.g., Npy1r) had mostly decreased expression. Genes for 27 potassium, 10 sodium, and 5 calcium ion channels were found to be differentially expressed. Nine genes in the cholesterol synthesis pathway had decreased expression, including Hmgcr, encoding the rate-limiting enzyme. Genes involved in the production of myelin also had decreased expression. CONCLUSIONS: The results demonstrate that binge alcohol drinking during adolescence produces developmental changes in the expression of key genes within the PAG; many of these changes point to increased susceptibility to pain, fear, and anxiety, which could contribute to excessive drinking to relieve these negative effects

Stress-response pathways are altered in the hippocampus of chronic alcoholics

The chronic high-level alcohol consumption seen in alcoholism leads to dramatic effects on the hippocampus, including decreased white matter, loss of oligodendrocytes and other glial cells, and inhibition of neurogenesis. Examining gene expression in post mortem hippocampal tissue from 20 alcoholics and 19 controls allowed us to detect differentially expressed genes that may play a role in the risk for alcoholism or whose expression is modified by chronic consumption of alcohol. We identified 639 named genes whose expression significantly differed between alcoholics and controls at a False Discovery Rate (FDR) ≤ 0.20; 52% of these genes differed by at least 1.2-fold. Differentially expressed genes included the glucocorticoid receptor and the related gene FK506 binding protein 5 (FKBP5), UDP glycosyltransferase 8 (UGT8), urea transporter (SLC14A1), zinc transporter (SLC39A10), Interleukin 1 receptor type 1 (IL1R1), thioredoxin interacting protein (TXNIP), and many metallothioneins. Pathways related to inflammation, hypoxia, and stress showed activation, and pathways that play roles in neurogenesis and myelination showed decreases. The cortisol pathway dysregulation and increased inflammation identified here are seen in other stress-related conditions such as depression and post-traumatic stress disorder and most likely play a role in addiction. Many of the detrimental effects on the hippocampus appear to be mediated through NF-κB signaling. Twenty-four of the differentially regulated genes were previously identified by genome-wide association studies of alcohol use disorders; this raises the potential interest of genes not normally associated with alcoholism, such as suppression of tumorigenicity 18 (ST18), BCL2-associated athanogene 3 (BAG3), and von Willebrand factor (VWF)

Asymptotically Optimal Encodings of Range Data Structures for Selection and Top-k Queries

Given an array A[1, n] of elements with a total order, we consider the problem of building a data structure that solves two queries: (a) selection queries receive a range [i, j] and an integer k and return the position of the kth largest element in A[i, j]; (b) top-k queries receive [i, j] and k and return the positions of the k largest elements in A[i, j]. These problems can be solved in optimal time, O(1 + lg k/ lg lg n) and O(k), respectively, using linear-space data structures. We provide the first study of the encoding data structures for the above problems, where A cannot be accessed at query time. Several applications are interested in the relative order of the entries of A, and their positions, rather their actual values, and thus we do not need to keep A at query time. In those cases, encodings save storage space: we first show that any encoding answering such queries requires n lg k − O(n + k lg k) bits of space; then, we design encodings using O(n lg k) bits, that is, asymptotically optimal up to constant factors, while preserving optimal query time.Peer-reviewedPost-prin