Search results for: bioconductor

Authors: Ruchi Yadav, Shivani Pandey, Prachi Srivastava

Abstract:

Microarrays provide a rich source of data on the molecular working of cells. Each microarray reports on the abundance of tens of thousands of mRNAs. Virtually every human disease is being studied using microarrays with the hope of finding the molecular mechanisms of disease. Bioinformatics analysis plays an important part of processing the information embedded in large-scale expression profiling studies and for laying the foundation for biological interpretation. A basic, yet challenging task in the analysis of microarray gene expression data is the identification of changes in gene expression that are associated with particular biological conditions. Careful statistical design and analysis are essential to improve the efficiency and reliability of microarray experiments throughout the data acquisition and analysis process. One of the most popular platforms for microarray analysis is Bioconductor, an open source and open development software project based on the R programming language. This paper describes specific procedures for conducting quality assessment, visualization and preprocessing of Affymetrix Gene Chip and also details the different bioconductor packages used to analyze affymetrix microarray data and describe the analysis and outcome of each plots.

Keywords: microarray analysis, R language, affymetrix visualization, bioconductor

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Authors: Quan Gu, Daniel Dimitrov

Abstract:

BingleSeq was developed as a shiny-based, intuitive, and comprehensive application that enables the analysis of single-Cell RNA-Sequencing count data. This was achieved via incorporating three state-of-the-art software packages for each type of RNA sequencing analysis, alongside functional annotation analysis and a way to assess the overlap of differential expression method results. At its current state, the functionality implemented within BingleSeq is comparable to that of other applications, also developed with the purpose of lowering the entry requirements to RNA Sequencing analyses. BingleSeq is available on GitHub and will be submitted to R/Bioconductor.

Keywords: bioinformatics, functional annotation analysis, single-cell RNA-sequencing, transcriptomics

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Authors: Jialin Ma, Jia Meng

Abstract:

Genomic features, the genome-based coordinates, are commonly used for the representation of biological features such as genes, RNA transcripts and transcription factor binding sites. For the analysis of RNA-related genomic features, such as RNA modification sites, a common task is to correlate these features with transcript components (5'UTR, CDS, 3'UTR) to explore their distribution characteristics in terms of transcriptomic coordinates, e.g., to examine whether a specific type of biological feature is enriched near transcription start sites. Existing approaches for performing these tasks involve the manipulation of a gene database, conversion from genome-based coordinate to transcript-based coordinate, and visualization methods that are capable of showing RNA transcript components and distribution of the features. These steps are complicated and time consuming, and this is especially true for researchers who are not familiar with relevant tools. To overcome this obstacle, we develop a dedicated web app TARF, which represents web toolkit for annotating RNA-related genomic features. TARF web tool intends to provide a web-based way to easily annotate and visualize RNA-related genomic features. Once a user has uploaded the features with BED format and specified a built-in transcript database or uploaded a customized gene database with GTF format, the tool could fulfill its three main functions. First, it adds annotation on gene and RNA transcript components. For every features provided by the user, the overlapping with RNA transcript components are identified, and the information is combined in one table which is available for copy and download. Summary statistics about ambiguous belongings are also carried out. Second, the tool provides a convenient visualization method of the features on single gene/transcript level. For the selected gene, the tool shows the features with gene model on genome-based view, and also maps the features to transcript-based coordinate and show the distribution against one single spliced RNA transcript. Third, a global transcriptomic view of the genomic features is generated utilizing the Guitar R/Bioconductor package. The distribution of features on RNA transcripts are normalized with respect to RNA transcript landmarks and the enrichment of the features on different RNA transcript components is demonstrated. We tested the newly developed TARF toolkit with 3 different types of genomics features related to chromatin H3K4me3, RNA N6-methyladenosine (m6A) and RNA 5-methylcytosine (m5C), which are obtained from ChIP-Seq, MeRIP-Seq and RNA BS-Seq data, respectively. TARF successfully revealed their respective distribution characteristics, i.e. H3K4me3, m6A and m5C are enriched near transcription starting sites, stop codons and 5’UTRs, respectively. Overall, TARF is a useful web toolkit for annotation and visualization of RNA-related genomic features, and should help simplify the analysis of various RNA-related genomic features, especially those related RNA modifications.

Keywords: RNA-related genomic features, annotation, visualization, web server

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Authors: Bianca Peterson, Tomasz J. Sańko, Carlos C. Bezuidenhout, Johnnie Van Den Berg

Abstract:

Busseola fusca (Fuller) (Lepidoptera: Noctuidae), the maize stem borer, is a major pest in sub-Saharan Africa. It causes economic damage to maize and sorghum crops and has evolved non-recessive resistance to genetically modified (GM) maize expressing the Cry1Ab insecticidal toxin. Since B. fusca is a non-model organism, very little genomic information is publicly available, and is limited to some cytochrome c oxidase I, cytochrome b, and microsatellite data. The biology of B. fusca is well-described, but still poorly understood. This, in combination with its larval-specific behavior, may pose problems for limiting the spread of current resistant B. fusca populations or preventing resistance evolution in other susceptible populations. As part of on-going research into resistance evolution, B. fusca larvae were collected from Bt and non-Bt maize in South Africa, followed by RNA isolation (15 specimens) and sequencing on the Illumina HiSeq 2500 platform. Quality of reads was assessed with FastQC, after which Trimmomatic was used to trim adapters and remove low quality, short reads. Trinity was used for the de novo assembly, whereas TransRate was used for assembly quality assessment. Transcript identification employed BLAST (BLASTn, BLASTp, and tBLASTx comparisons), for which two libraries (nucleotide and protein) were created from 3.27 million lepidopteran sequences. Several transcripts that have previously been implicated in Cry toxin resistance was identified for B. fusca. These included aminopeptidase N, cadherin, alkaline phosphatase, ATP-binding cassette transporter proteins, and mitogen-activated protein kinase. MEGA7 was used to align these transcripts to reference sequences from Lepidoptera to detect mutations that might potentially be contributing to Cry toxin resistance in this pest. RSEM and Bioconductor were used to perform differential gene expression analysis on groups of B. fusca larvae challenged and unchallenged with the Cry1Ab toxin. Pairwise expression comparisons of transcripts that were at least 16-fold expressed at a false-discovery corrected statistical significance (p) ≤ 0.001 were extracted and visualized in a hierarchically clustered heatmap using R. A total of 329,194 transcripts with an N50 of 1,019 bp were generated from the over 167.5 million high-quality paired-end reads. Furthermore, 110 transcripts were over 10 kbp long, of which the largest one was 29,395 bp. BLAST comparisons resulted in identification of 157,099 (47.72%) transcripts, among which only 3,718 (2.37%) were identified as Cry toxin receptors from lepidopteran insects. According to transcript expression profiles, transcripts were grouped into three subclusters according to the similarity of their expression patterns. Several immune-related transcripts (pathogen recognition receptors, antimicrobial peptides, and inhibitors) were up-regulated in the larvae feeding on Bt maize, indicating an enhanced immune status in response to toxin exposure. Above all, extremely up-regulated arylphorin genes suggest that enhanced epithelial healing is one of the resistance mechanisms employed by B. fusca larvae against the Cry1Ab toxin. This study is the first to provide a resource base and some insights into a potential mechanism of Cry1Ab toxin resistance in B. fusca. Transcriptomic data generated in this study allows identification of genes that can be targeted by biotechnological improvements of GM crops.

Keywords: epithelial healing, Lepidoptera, resistance, transcriptome

Procedia PDF Downloads 163