Transcriptomic Profiling Using Next Generation Sequencing - Advances, Advantages, and Challenges

Transcriptome, the functional element of the genome, is comprised of different kinds of RNA molecules such as mRNA, miRNA, ncRNA, rRNA, and tRNA to name a few. Each of these RNA molecules plays a vital role in the physiological response, and understanding the regulation of these molecules is extremely critical for the better understanding of the functional genome. RNA Sequencing (RNASeq) is one of the latest techniques applied to study genome-wide transcriptome characterization and profiling using high-throughput sequenced data. As compared to array-based methods, RNASeq provides in-depth and more precise information on transcriptome characterization and quantification. Based upon availability of reference genome, transcriptome assembly can be reference-guided or de novo. Once transcripts are assembled, downstream analysis such as expression profiling, gene ontology, and pathway enrichment analyses can give more insight into gene regulation. This chapter describes the significance of RNASeq study over array-based traditional methods, approach to analyze RNASeq data, available methods and tools, challenges associated with the data analysis, application areas, some of the recent advancement made in the area of transcriptome study and its application

Conservation Patterns of HIV-1 RT Connection and RNase H Domains: Identification of New Mutations in NRTI-Treated Patients

Background: Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. Methods and Findings: We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naıve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher’s exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. Conclusions: This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions

FusionHub: A unified web platform for annotation and visualization of gene fusion events in human cancer.

Gene fusion is a chromosomal rearrangement event which plays a significant role in cancer due to the oncogenic potential of the chimeric protein generated through fusions. At present many databases are available in public domain which provides detailed information about known gene fusion events and their functional role. Existing gene fusion detection tools, based on analysis of transcriptomics data usually report a large number of fusion genes as potential candidates, which could be either known or novel or false positives. Manual annotation of these putative genes is indeed time-consuming. We have developed a web platform FusionHub, which acts as integrated search engine interfacing various fusion gene databases and simplifies large scale annotation of fusion genes in a seamless way. In addition, FusionHub provides three ways of visualizing fusion events: circular view, domain architecture view and network view. Design of potential siRNA molecules through ensemble method is another utility integrated in FusionHub that could aid in siRNA-based targeted therapy. FusionHub is freely available at https://fusionhub.persistent.co.in

Additional file 13: of Elucidating mechanistic insights into drug action for atopic dermatitis: a systems biology approach

Figure S10. Expression profile of skin barrier proteins in BM and PC samples. (a) Expression profile of genes that are upregulated in both BM and PC in order to restore barrier functions; (b) Expression profile of skin barrier genes that show treatment specific difference in their expressions. (TIFF 8378 kb

FusionHub server implementation with its three modules, FusionSearch, FusionView and siRNAdesign.

<p>The numbers in parenthesis in FusionSearch module correspond to number of fusion genes in that dataset. FusionDatabase consists of a total of 150699 fusion list, pooled from the 24 datasets listed in FusionSearch module.</p

Description of 24 fusion gene datasets included in FusionHub server.

<p>Description of 24 fusion gene datasets included in FusionHub server.</p

Annotations obtained for CCDC6—RET gene fusion from various databases.

<p>Only few annotations are shown here. Full annotation can be browsed at <a href="https://fusionhub.persistent.co.in/out/Example1/Individual/CCDC6—RET.html" target="_blank">https://fusionhub.persistent.co.in/out/Example1/Individual/CCDC6—RET.html</a>.</p

Result of siRNA prediction for RBX1 gene.

<p>Result of siRNA prediction for RBX1 gene.</p

FusionHub: A unified web platform for annotation and visualization of gene fusion events in human cancer - Fig 4

<p>The different views available in FusionView module, circular view (A), domain architecture view (B) and network view (C). Circular view (A) shows fusion events for 6 input genes provided as input. Domain view shows three different ways by which CCDC6—RET fusion gene is formed (B). Network view shows the fusion gene network involving CCDC6 gene and its fusion interaction partners.</p

Summary page obtained after running FusionSearch module for CCDC6—RET gene.

<p>The result page can be browsed at <a href="https://fusionhub.persistent.co.in/cgi-bin/result_fetch_fusion.php?ID=Example1" target="_blank">https://fusionhub.persistent.co.in/cgi-bin/result_fetch_fusion.php?ID=Example1</a>.</p