Sanjit Pandey

A new rhesus macaque assembly and annotation for next-generation sequencing analyses

BackgroundThe rhesus macaque (Macaca mulatta) is a key species for advancing biomedical research. Like all draft mammalian genomes, the draft rhesus assembly (rheMac2) has gaps, sequencing errors and misassemblies that have prevented automated annotation pipelines from functioning correctly. Another rhesus macaque assembly, CR_1.0, is also available but is substantially more fragmented than rheMac2 with smaller contigs and scaffolds. Annotations for these two assemblies are limited in completeness and accuracy. High quality assembly and annotation files are required for a wide range of studies including expression, genetic and evolutionary analyses.ResultsWe report a new de novo assembly of the rhesus macaque genome (MacaM) that incorporates both the original Sanger sequences used to assemble rheMac2 and new Illumina sequences from the same animal. MacaM has a weighted average (N50) contig size of 64 kilobases, more than twice the size of the rheMac2 assembly and almost five times the size of the CR_1.0 assembly. The MacaM chromosome assembly incorporates information from previously unutilized mapping data and preliminary annotation of scaffolds. Independent assessment of the assemblies using Ion Torrent read alignments indicates that MacaM is more complete and accurate than rheMac2 and CR_1.0. We assembled messenger RNA sequences from several rhesus tissues into transcripts which allowed us to identify a total of 11,712 complete proteins representing 9,524 distinct genes. Using a combination of our assembled rhesus macaque transcripts and human transcripts, we annotated 18,757 transcripts and 16,050 genes with complete coding sequences in the MacaM assembly. Further, we demonstrate that the new annotations provide greatly improved accuracy as compared to the current annotations of rheMac2. Finally, we show that the MacaM genome provides an accurate resource for alignment of reads produced by RNA sequence expression studies.ConclusionsThe MacaM assembly and annotation files provide a substantially more complete and accurate representation of the rhesus macaque genome than rheMac2 or CR_1.0 and will serve as an important resource for investigators conducting next-generation sequencing studies with nonhuman primates.ReviewersThis article was reviewed by Dr. Lutz Walter, Dr. Soojin Yi and Dr. Kateryna Makova.

Mammalian Alteration/Deficiency in Activation 3 (Ada3) Is Essential for Embryonic Development and Cell Cycle Progression

Background: Ada3 is a core component of HAT containing coactivator complexes. Results: Germline deletion of Ada3 is embryonic lethal, and cell deletion leads to abnormal cell cycle progression. Conclusion: Ada3 is a critical protein at organismic and cellular level. Significance: This study describes a novel role of Ada3, a component of HAT complexes, as a critical regulator of cell survival. Ada3 protein is an essential component of histone acetyl transferase containing coactivator complexes conserved from yeast to human. We show here that germline deletion of Ada3 in mouse is embryonic lethal, and adenovirus-Cre mediated conditional deletion of Ada3 in Ada3FL/FL mouse embryonic fibroblasts leads to a severe proliferation defect which was rescued by ectopic expression of human Ada3. A delay in G1 to S phase of cell cycle was also seen that was due to accumulation of Cdk inhibitor p27 which was an indirect effect of c-myc gene transcription control by Ada3. We further showed that this defect could be partially reverted by knocking down p27. Additionally, drastic changes in global histone acetylation and changes in global gene expression were observed in microarray analyses upon loss of Ada3. Lastly, formation of abnormal nuclei, mitotic defects and delay in G2/M to G1 transition was seen in Ada3 deleted cells. Taken together, we provide evidence for a critical role of Ada3 in embryogenesis and cell cycle progression as an essential component of HAT complex.

ngLOC: software and web server for predicting protein subcellular localization in prokaryotes and eukaryotes

BackgroundUnderstanding protein subcellular localization is a necessary component toward understanding the overall function of a protein. Numerous computational methods have been published over the past decade, with varying degrees of success. Despite the large number of published methods in this area, only a small fraction of them are available for researchers to use in their own studies. Of those that are available, many are limited by predicting only a small number of organelles in the cell. Additionally, the majority of methods predict only a single location for a sequence, even though it is known that a large fraction of the proteins in eukaryotic species shuttle between locations to carry out their function.FindingsWe present a software package and a web server for predicting the subcellular localization of protein sequences based on the ngLOC method. ngLOC is an n-gram-based Bayesian classifier that predicts subcellular localization of proteins both in prokaryotes and eukaryotes. The overall prediction accuracy varies from 89.8% to 91.4% across species. This program can predict 11 distinct locations each in plant and animal species. ngLOC also predicts 4 and 5 distinct locations on gram-positive and gram-negative bacterial datasets, respectively.ConclusionsngLOC is a generic method that can be trained by data from a variety of species or classes for predicting protein subcellular localization. The standalone software is freely available for academic use under GNU GPL, and the ngLOC web server is also accessible at http://ngloc.unmc.edu.

LocSigDB: a database of protein localization signals

LocSigDB (http://genome.unmc.edu/LocSigDB/) is a manually curated database of experimental protein localization signals for eight distinct subcellular locations; primarily in a eukaryotic cell with brief coverage of bacterial proteins. Proteins must be localized at their appropriate subcellular compartment to perform their desired function. Mislocalization of proteins to unintended locations is a causative factor for many human diseases; therefore, collection of known sorting signals will help support many important areas of biomedical research. By performing an extensive literature study, we compiled a collection of 533 experimentally determined localization signals, along with the proteins that harbor such signals. Each signal in the LocSigDB is annotated with its localization, source, PubMed references and is linked to the proteins in UniProt database along with the organism information that contain the same amino acid pattern as the given signal. From LocSigDB webserver, users can download the whole database or browse/search for data using an intuitive query interface. To date, LocSigDB is the most comprehensive compendium of protein localization signals for eight distinct subcellular locations. Database URL: http://genome.unmc.edu/LocSigDB/

Implications of TGFβ on Transcriptome and Cellular Biofunctions of Palatal Mesenchyme

Development of the palate comprises sequential stages of growth, elevation, and fusion of the palatal shelves. The mesenchymal component of palates plays a major role in early phases of palatogenesis, such as growth and elevation. Failure in these steps may result in cleft palate, the second most common birth defect in the world. These early stages of palatogenesis require precise and chronological orchestration of key physiological processes, such as growth, proliferation, differentiation, migration, and apoptosis. There is compelling evidence for the vital role of TGFβ-mediated regulation of palate development. We hypothesized that the isoforms of TGFβ regulate different cellular biofunctions of the palatal mesenchyme to various extents. Human embryonic palatal mesenchyme (HEPM) cells were treated with TGFβ1, β2, and β3 for microarray-based gene expression studies in order to identify the roles of TGFβ in the transcriptome of the palatal mesenchyme. Following normalization and modeling of 28,869 human genes, 566 transcripts were detected as differentially expressed in TGFβ-treated HEPM cells. Out of these altered transcripts, 234 of them were clustered in cellular biofunctions, including growth and proliferation, development, morphology, movement, cell cycle, and apoptosis. Biological interpretation and network analysis of the genes active in cellular biofunctions were performed using IPA. Among the differentially expressed genes, 11 of them are known to be crucial for palatogenesis (EDN1, INHBA, LHX8, PDGFC, PIGA, RUNX1, SNAI1, SMAD3, TGFβ1, TGFβ2, and TGFβR1). These genes were used for a merged interaction network with cellular behaviors. Overall, we have determined that more than 2% of human transcripts were differentially expressed in response to TGFβ treatment in HEPM cells. Our results suggest that both TGFβ1 and TGFβ2 orchestrate major cellular biofunctions within the palatal mesenchyme in vitro by regulating expression of 234 genes.

SWATH-MS proteome profiling data comparison of DJ-1, Parkin, and PINK1 knockout rat striatal mitochondria

This article reports changes in the striatal non-synaptic mitochondrial proteome of DJ-1, Parkin, and PINK1 knockout (KO) rats at 3 months of age. DJ-1, Parkin, and PINK1 mutations cause autosomal-recessive parkinsonism. It is thought that loss of function of these proteins contributes to the onset and pathogenesis of Parkinson׳s disease (PD). As DJ-1, Parkin, and PINK1 have functions in the regulation of mitochondria, the dataset generated here highlights protein expression changes, which can be helpful for understanding pathological mitochondrial alterations. In total, 1281 proteins were quantified and 25, 37, and 15 proteins were found to exhibit differential expression due to DJ-1, Parkin, and PINK1 deficiency, respectively. All quantification can be found in the supplemental table and can be searched online at http://genome.unmc.edu/mitorat/index.html. Further, mitochondrial respiration was measured to evaluate mitochondrial function in the striatum of DJ-1, Parkin, and PINK1 KO rats, which was significantly changed only in the DJ-1 KOs.

Changes in ovarian protein expression during primordial follicle formation in the hamster

Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and their interaction with the oocytes.

The National NeuroAIDS Tissue Consortium (NNTC) Database: an integrated database for HIV-related studies

We herein present the National NeuroAIDS Tissue Consortium-Data Coordinating Center (NNTC-DCC) database, which is the only available database for neuroAIDS studies that contains data in an integrated, standardized form. This database has been created in conjunction with the NNTC, which provides human tissue and biofluid samples to individual researchers to conduct studies focused on neuroAIDS. The database contains experimental datasets from 1206 subjects for the following categories (which are further broken down into subcategories): gene expression, genotype, proteins, endo-exo-chemicals, morphometrics and other (miscellaneous) data. The database also contains a wide variety of downloadable data and metadata for 95 HIV-related studies covering 170 assays from 61 principal investigators. The data represent 76 tissue types, 25 measurement types, and 38 technology types, and reaches a total of 33 017 407 data points. We used the ISA platform to create the database and develop a searchable web interface for querying the data. A gene search tool is also available, which searches for NCBI GEO datasets associated with selected genes. The database is manually curated with many user-friendly features, and is cross-linked to the NCBI, HUGO and PubMed databases. A free registration is required for qualified users to access the database. Database URL: http://nntc-dcc.unmc.edu

Abstract B12: Genomic and epigenomic characterization of global DNA hypomethylation in human epithelial ovarian cancer

Background: Epithelial ovarian cancer (EOC), and other solid tumors, contain a variable proportion of tumors characterized by global DNA hypomethylation. This phenotype is known to involve loss of DNA methylation at repetitive elements (RE) and at single copy gene promoters, including cancer-germline (a.k.a. cancer-testis) antigen genes. Global DNA hypomethylation in EOC coincides with tumor progression and reduced survival in EOC patients. Despite the clinical importance of global DNA hypomethylation, we currently have a limited understanding of the origin, the targets, and the consequences of this EOC phenotype. Objectives: The purpose of this study was to: i) examine global gene expression in globally hypomethylated EOC, ii) to identify the molecular pathways deregulated in these tumors, iii) to discover and validate novel and clinically relevant targets of DNA hypomethylation in EOC, iv) to conduct DNA methylome analysis to more precisely define the genomic locations effected by global DNA hypomethylation in EOC, and v) to determine whether global DNA hypomethylation impact RE gene expression in EOC. Methods: Global methylation status in EOC was determined using quantitative bisulfite pyrosequencing of the LINE1 RE. Affymetrix microarrays, RT-qPCR, and total RNAseq were used to measure gene expression in normal ovary (NO), LINE1 hypomethylated EOC, and LINE1 hypermethylated (i.e. normally methylated) EOC. Pathway analysis was conducted on differentially expressed genes (DEG) between NO and EOC and between the two EOC groups. Illumina Infinium 450K arrays and Agilent Sure Select methyl-seq were used to examine the DNA methylome of NO and EOC. Quantitative sodium bisulfite pyrosequencing was used to determine locus-specific DNA methylation in NO and EOC. Results: Global mRNA expression was distinct in LINE1 hypomethylated EOC, with ~70% of DEG upregulated, implicating DNA hypomethylation in gene activation. The most significantly altered pathway in LINE1 hypomethylated EOC was cell cycle, implicating enhanced proliferation in this phenotype. Cancer germline genes not previously known to be regulated by DNA methylation in EOC, including CT45 and PRAME, were identified and characterized. DNA methylome analysis of LINE1 hypomethylated EOC indicated that hypomethylation is not evenly dispersed across the genome, but rather is regionally localized, including at nuclear lamina associated domains (LAD). Preliminary analysis of RNAseq data indicated that specific RE are overexpressed in LINE1 hypomethylated tumors. Conclusions: Pathway analysis suggests that rapid cellular proliferation coupled with inherent inefficiencies of maintenance DNA methylation at specific genomic locations may contribute to global DNA hypomethylation in EOC. DNA hypomethylation is linked to gene activation in EOC, and important targets of this defect are cancer germline genes, which are likely to contribute to oncogenesis but may also be targeted by immunotherapy. Global DNA hypomethylation appears to be concentrated at specific genomic locations including LADs, and is connected not only to RE hypomethylation, but also to enhanced RE expression. Higher-order nuclear changes and enhanced RE expression are potential mechanisms driving genomic instability and/or poor prognosis in EOC patients displaying global DNA hypomethylation. Citation Format: Wa Zhang, David Klinkebiel, Sanjit Pandey, Dan Wang, Song Liu, Chittibabu Guda, Kunle Odunsi, Adam R. Karpf. Genomic and epigenomic characterization of global DNA hypomethylation in human epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B12.