Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes

© 2017 Wong et al.; Published by Cold Spring Harbor Laboratory Press. Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted noncoding RNAs to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes

Fine mapping and sequence analysis reveal a promising candidate gene encoding a novel NB-ARC domain derived from wild rice (Oryza officinalis) that confers bacterial blight resistance

Bacterial blight disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious constraints in rice production. The most sustainable strategy to combat the disease is the deployment of host plant resistance. Earlier, we identified an introgression line, IR 75084-15-3-B-B, derived from Oryza officinalis possessing broad-spectrum resistance against Xoo. In order to understand the inheritance of resistance in the O. officinalis accession and identify genomic region(s) associated with resistance, a recombinant inbred line (RIL) mapping population was developed from the cross Samba Mahsuri (susceptible to bacterial blight) × IR 75084-15-3-B-B (resistant to bacterial blight). The F2 population derived from the cross segregated in a phenotypic ratio of 3: 1 (resistant susceptible) implying that resistance in IR 75084-15-3-B-B is controlled by a single dominant gene/quantitative trait locus (QTL). In the F7 generation, a set of 47 homozygous resistant lines and 47 homozygous susceptible lines was used to study the association between phenotypic data obtained through screening with Xoo and genotypic data obtained through analysis of 7K rice single-nucleotide polymorphism (SNP) chip. Through composite interval mapping, a major locus was detected in the midst of two flanking SNP markers, viz., Chr11.27817978 and Chr11.27994133, on chromosome 11L with a logarithm of the odds (LOD) score of 10.21 and 35.93% of phenotypic variation, and the locus has been named Xa48t. In silico search in the genomic region between the two markers flanking Xa48t identified 10 putatively expressed genes located in the region of interest. The quantitative expression and DNA sequence analysis of these genes from contrasting parents identified the Os11g0687900 encoding an NB-ARC domain-containing protein as the most promising gene associated with resistance. Interestingly, a 16-bp insertion was noticed in the untranslated region (UTR) of the gene in the resistant parent, IR 75084-15-3-B-B, which was absent in Samba Mahsuri. The association of Os11g0687900 with resistance phenotype was further established by sequence-based DNA marker analysis in the RIL population. A co-segregating PCR-based INDEL marker, Marker_Xa48, has been developed for use in the marker-assisted breeding of Xa48t

Surgery, Octreotide, Temozolomide, Bevacizumab, Radiotherapy, and Pegvisomant Treatment of an AIP Mutation-Positive Child

UK India Education Research Initiative and the British Council (75-2014; to P.D.)Council of Scientific and Industrial Research, University Grants Commission, for financial support (2061330632; to A.R.).Medical Research Council (MR/M018539/1; to M.K

Chronic shisha exposure alters phosphoproteome of oral keratinocytes

Shisha smoking has been epidemiologically linked to oral cancer. However, few studies have investigated the pathobiology of shisha-induced cellular transformation. We studied the effects of chronic shisha exposure (8 months) in an in vitro model using immortalized, non-neoplastic oral keratinocytes (OKF6/TERT1). Quantitative proteomic and phosphoproteomic analyses were performed on OKF6/TERT1 cells treated with shisha extract for a period of 8 months. Pathway analysis was carried out to identify significantly enriched biological processes in shisha-treated cells. Chronic shisha exposure resulted in increased cell scattering phenomenon in OKF6/TERT1 cells. Data analysis revealed differential phosphorylation of 164 peptides (fold change >= 1.5,

Data on whole genome sequencing of extrapulmonary tuberculosis clinical isolates from India

This article describes the whole genome sequencing data from 5 extrapulmonary tuberculosis clinical isolates. The whole genome sequencing was carried out on Illumina MiSeq platform to identify single nucleotide variations (SNVs) associated with drug resistance. A total of 214 SNVs in the coding and promoter regions were identified in the whole genome sequencing analysis. Among the identified SNVs, 18 SNVs were identified in genes known to be associated with first and second line drug resistance. The data is related to the research article “Whole genome sequencing of Mycobacterium tuberculosis isolates from extrapulmonary sites” (Sharma et al., 2017) [1]

Delineating miRNA profile induced by chewing tobacco in oral keratinocytes

The major established etiologic risk factor for oral cancer is tobacco (chewed, smoked and snuffed forms). Chewing form of tobacco is predominantly used in India making it the leading cause of oral cancer. Despite being one of the leading causes of oral cancer, the molecular alterations induced by chewing tobacco remains largely unclear. Carcinogenic effect of chewing tobacco is through chronic and not acute exposure. To understand the molecular alterations induced by chewing tobacco, we developed a cell line model where non-neoplastic oral keratinocytes were chronically exposed to chewing tobacco for a period of 6 months. This resulted in increased cellular proliferation and invasive ability of normal oral keratinocytes. Using this cellular model we studied the differential expression of miRNAs associated with chewing tobacco and the altered signaling pathways through which the aberrantly expressed miRNAs affect tumorigenesis. miRNA sequencing  was carried out using Illumina HiSeq 2500 platform  which resulted in the identification of 427 annotated miRNAs of which 10 were significantly dysregulated (≥ 4 fold; p-value ≤ 0.05) in tobacco exposed cells compared to untreated parental cells. To study the altered signaling in oral keratinocytes chronically exposed to chewing tobacco, we employed quantitative proteomics to characterize the dysregulated proteins. Integration of miRNA sequencing data with proteomic data resulted in identification of 36 proven protein targets which (≥1.5 fold; p-value ≤ 0.05) showed expression correlation with the 10 significantly dysregulated miRNAs. Pathway analysis of the dysregulated targets revealed enrichment of interferon signaling and mRNA processing related pathways in the chewing tobacco exposed cells. In addition, we also identified 6 novel miRNA in oral keratinocytes chronically exposed to chewing tobacco extract. Our study provides a framework to understand the oncogenic transformation induced by chromic tobacco exposure in normal oral keratinocytes

Cigarette smoke induces metabolic reprogramming in lung cells

Cigarette smoking remains the leading cause of non-small cell lung carcinoma. Studies involving acute exposure of smoke on lung cells revealed induction of pre- cancerous state in lung cells. Recently few studies have reported the chronic effect of cigarette smoke in inducing cellular transformation. Yet no systemic study has been performed to understand the molecular alterations in lung cells due to cigarette smoke. Hence it is both important and necessary to study the chronic effect of cigarette smoke in a temporal setting to understand the molecular alterations. In this study, we carried out TMT based proteomic profiling of lung cells which were exposed to cigarette smoke condensate (CSC) for upto 12 months. We identified 2621 proteins in total, of which 145, 114, 87, 169 and 671 proteins were differentially expressed (p<0.05, 1.5 fold) in 2nd, 4th, 6th, 8th and 12th month respectively.Pathway analysis revealed enrichment of xenobiotic metabolism signaling for the first 8 months of smoke treatment, whereas continued exposure of smoke for 12 months revealed mitochondrial reprogramming in cells which includes dysregulation of oxidative phosphorylation machinery leading to enhanced reactive oxygen species and higher expression of enzymes involved in tricarboxylic acid cycle (TCA). In addition, chronic exposure of smoke led to overexpression of enzymes involved in glutamine metabolism, fatty acid degradation and lactate synthesis. This could possibly explain the availability of alternative source of carbon in TCA cycle apart from glycolytic pyruvate. Our data indicates that chronic exposure to cigarette smoke induces metabolic transformation in cells to support growth and survival

miRNA and proteomic dysregulation in non-small cell lung cancer in response to cigarette smoke

Dysregulation of miRNAs is well associated with the development of non-small cell lung cancer (NSCLC). It is imperative that dysregulation of miRNAs by cigarette smoke will affect the expression of their targets, either leading to the activation of oncoproteins or suppression of tumor suppressor proteins. In this study, we have carried out miRNA sequencing and SILAC-based proteomics analysis of H358 cells chronically exposed to cigarette smoke condensate. miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (fold change ≥ 4, p-value ≤ 0.05) in H358-smoke exposed cells. Proteomic analysis of the smoke exposed cells compared to the parental cells resulted in the quantification of 2,396 proteins, of which 681 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins involved in biological processes driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway analysis using Ingenuity Pathway Analysis (IPA) revealed activation of ERK/MAPK and integrin signaling and repression of RhoGDI signaling in H358 smoke exposed cells. We also identified 5 novel miRNA in H358 smoke exposed cells using unassigned reads of small RNA-Seq dataset. In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential targets and/or molecular markers in NSCLC especially in smokers

Cigarette smoke and chewing tobacco alter expression of different sets of miRNAs in oral keratinocytes.

Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco

Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells

Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers