BOL: Related items

BioJupies: Automatically Generates RNA-seq Data Analysis Notebooks

Rahul Nayak — Sun, 20 Dec 2020 11:43:45 -0600

With BioJupies you can produce in seconds a customized, reusable, and interactive report from your own raw or processed RNA-seq data through a simple user interface

BioJupies now supports user accounts! Sign in from the top right corner of the page for access to unlimited private notebooks, RNA-seq datasets and alignment jobs.

Address of the bookmark: https://amp.pharm.mssm.edu/biojupies/

RNA-seq workflow: gene-level exploratory analysis and differential expression

Jit — Sat, 09 Oct 2021 07:59:23 -0500

Here we walk through an end-to-end gene-level RNA-seq differential expression workflow using Bioconductor packages. We will start from the FASTQ files, show how these were quantified to the reference transcripts, and prepare gene-level count datasets for downstream analysis. We will perform exploratory data analysis (EDA) for quality assessment and to explore the relationship between samples, perform differential gene expression analysis, and visually explore the results.

Address of the bookmark: http://master.bioconductor.org/packages/release/workflows/vignettes/rnaseqGene/inst/doc/rnaseqGene.html

deepTools

Martin Jones — Sat, 08 Nov 2014 15:02:08 -0600

deepTools addresses the challenge of handling the large amounts of data that are now routinely generated from DNA sequencing centers. To do so, deepTools contains useful modules to process the mapped reads data to create coverage files in standard bedGraph and bigWig file formats. By doing so, deepTools allows the creation of normalized coverage files or the comparison between two files (for example, treatment and control). Finally, using such normalized and standardized files, multiple visualizations can be created to identify enrichments with functional annotations of the genome.

Publicaton: http://nar.oxfordjournals.org/content/early/2014/05/05/nar.gku365.full

Source Code and Wiki: https://github.com/fidelram/deepTools/wiki

Galaxy Tool Shed repository: http://toolshed.g2.bx.psu.edu/view/bgruening/deeptools

and example Galaxy workflows: http://toolshed.g2.bx.psu.edu/view/bgruening/deeptools_workflows

kallisto: a program for quantifying abundances of transcripts from bulk and single-cell RNA-Seq data

Jit — Mon, 07 Jan 2019 10:35:14 -0600

kallisto is a program for quantifying abundances of transcripts from bulk and single-cell RNA-Seq data, or more generally of target sequences using high-throughput sequencing reads. It is based on the novel idea of pseudoalignment for rapidly determining the compatibility of reads with targets, without the need for alignment. On benchmarks with standard RNA-Seq data, kallisto can quantify 30 million human reads in less than 3 minutes on a Mac desktop computer using only the read sequences and a transcriptome index that itself takes less than 10 minutes to build. Pseudoalignment of reads preserves the key information needed for quantification, and kallisto is therefore not only fast, but also as accurate as existing quantification tools. In fact, because the pseudoalignment procedure is robust to errors in the reads, in many benchmarks kallisto significantly outperforms existing tools. kallisto is described in detail in:

Nicolas L Bray, Harold Pimentel, Páll Melsted and Lior Pachter, Near-optimal probabilistic RNA-seq quantification, Nature Biotechnology 34, 525–527 (2016), doi:10.1038/nbt.3519

Address of the bookmark: https://pachterlab.github.io/kallisto/about

RNA-Seq Analysis: A Guide for Bioinformaticians

LEGE — Sat, 07 Dec 2024 22:22:24 -0600

RNA sequencing (RNA-Seq) has revolutionized transcriptomics, offering unprecedented insights into gene expression, splicing, and transcript diversity. For bioinformaticians, RNA-Seq analysis is a gateway to exploring the complexity of RNA biology and its implications in health and disease. This blog post provides an overview of RNA-Seq analysis, key computational steps, and tools for bioinformaticians eager to delve into this powerful technique.

What is RNA-Seq?

RNA-Seq is a next-generation sequencing (NGS) technology used to study the transcriptome—the complete set of RNA molecules in a cell. It quantifies gene expression, detects novel transcripts, and captures alternative splicing events with high sensitivity and resolution.

Workflow for RNA-Seq Analysis

RNA-Seq analysis involves several stages, each requiring computational tools and expertise.

1. Experimental Design and Data Acquisition

Before diving into analysis, bioinformaticians should consider:

Biological Replicates: Ensure statistical power to detect meaningful differences.
Sequencing Depth: Align sequencing depth to study objectives (e.g., higher depth for low-abundance transcripts).
Paired-End vs. Single-End: Paired-end sequencing provides more detailed information on transcript structure.

Once sequencing is complete, raw data is provided in FASTQ format, containing sequence reads and quality scores.

2. Quality Control and Preprocessing

Quality control (QC) ensures data integrity. Tools such as FastQC evaluate metrics like base quality, GC content, and adapter contamination.

Preprocessing Steps:

Trimming: Tools like Trimmomatic or Cutadapt remove low-quality bases and adapter sequences.
Filtering: Discard reads below a certain quality threshold or length.

3. Read Alignment

Reads are mapped to a reference genome or transcriptome to determine their origin. Alignment tools include:

HISAT2: Handles large genomes efficiently and supports spliced alignments.
STAR: High-speed aligner optimized for RNA-Seq.
Bowtie2: Suitable for short-read alignment.

Output: A SAM/BAM file containing aligned reads.

4. Transcript Assembly and Quantification

This step involves identifying transcripts and quantifying their expression levels. Tools used include:

StringTie: Assembles and quantifies transcripts from aligned reads.
Salmon/Kallisto: Perform pseudo-alignment for rapid and accurate quantification.

Expression levels are typically measured as TPM (transcripts per million) or FPKM (fragments per kilobase of transcript per million mapped reads).

5. Differential Expression Analysis

To identify genes with altered expression between conditions, bioinformaticians use tools such as:

DESeq2: Accounts for data normalization and variability.
edgeR: Handles overdispersed count data efficiently.
Limma-voom: Combines linear modeling with RNA-Seq count data.

The output includes a list of differentially expressed genes (DEGs) with statistical significance and fold-change values.

6. Functional Annotation and Pathway Analysis

Understanding the biological significance of DEGs involves:

Gene Ontology (GO) Analysis: Tools like DAVID or clusterProfiler categorize genes based on their biological functions.
Pathway Enrichment Analysis: Identifies pathways enriched in DEGs using tools like KEGG, Reactome, or GSEA.

7. Visualization

Visualizing results enhances interpretability. Common visualizations include:

Heatmaps: Show expression patterns across samples (e.g., pheatmap).
Volcano Plots: Highlight significant DEGs (e.g., ggplot2).
PCA/UMAP: Assess sample clustering and variability (e.g., Seurat).

Challenges in RNA-Seq Analysis

Batch Effects: Technical variability can confound biological signals. Combat this with normalization techniques or batch-correction tools like ComBat.
Low-Quality Samples: Poor-quality RNA impacts downstream analyses.
Computational Complexity: RNA-Seq generates massive datasets, requiring robust computing resources and optimized pipelines.

Key Tools and Resources

Bioconductor: A treasure trove of R packages for RNA-Seq analysis.
Galaxy: A web-based platform for running RNA-Seq workflows.
Nextflow/Snakemake: Workflow management tools to streamline analyses.

Applications of RNA-Seq

RNA-Seq is used in diverse research areas, including:

Cancer Transcriptomics: Identifying tumor-specific expression profiles.
Developmental Biology: Studying dynamic transcriptome changes.
Drug Discovery: Screening genes modulated by therapeutic compounds.

Conclusion

RNA-Seq analysis is a cornerstone of modern transcriptomics, offering bioinformaticians a versatile toolkit for unraveling gene expression and regulation. Mastering RNA-Seq workflows and tools empowers researchers to transform raw sequencing data into biological discoveries.

Whether you’re investigating disease mechanisms, exploring cellular pathways, or developing new therapeutics, RNA-Seq is a powerful ally in your bioinformatics arsenal.

Webinar on RNA-Seq Data Analysis on 28 Feb 2018

Strand — Thu, 22 Feb 2018 06:38:48 -0600

Strand NGS is a biologist friendly NGS analysis tool that allows biologists to analyze their data using a very intuitive workflow for the analysis and visualization of RNA-Seq data. This webinar will give an overview of the workflow which includes Transcriptome/ Genome alignment, Differential expression analysis, Splicing events and gene fusion detection. Strand NGS also supports novel discovery like identification of novel genes, exons and novel splice junctions.
We will highlight the use of Strand NGS features such as PCA, sample correlation, clustering, Venn diagrams, CVA, UMI support and elastic genome browser used in RNA-Seq workflow that supports large scale RNA-Seq data analysis too. The tool also supports biological contextualization on the set of interesting genes from the data by allowing downstream analysis such as GO and pathway analysis. The product has an option to create pipelines for time consuming jobs which automates analysis and leaves more time for end data interpretation. This webinar will give an overview of the features in the RNA-Seq data analysis workflow in Strand NGS.

Details:
Session 1: 28 Feb 2018, 9 AM CET
Session 2: 28 Feb 2018, 8 AM PST
Register here: http://www.strand-ngs.com/webinar_registration

About Speaker:

Dr. Suman Kapoor, Manager- Application Science at Strand Life Sciences, has over 11 years experience in molecular biology, next-generation sequencing based testing, clinical genomics, and personalized medicine for disease management and prenatal testing. Dr. Suman holds a Ph.D in Molecular and Cell Biology from Indian Institute of Science, Bangalore. Prior to joining Strand NGS team, Suman has worked extensively on protein synthesis in eubacteria and has experience working in CAP and NABL accredited lab validating and interpreting NGS based diagnostic tests.

Sequencing Solutions to World Health

Rahul Agarwal — Thu, 29 Aug 2013 15:05:35 -0500

"New technology that quickly, easily and economically reveals the genomes of viruses and pathogens transforms public health and medicine."

Source: Life technologies

Address of the bookmark: http://www.lifetechnologies.com/global/en/home/communities-social/blog/blogs/sequencing-solutions-to-world-health.html?cid=social_blogseries_20130829_11098264

Genome Browsers

Rahul Agarwal — Fri, 16 Aug 2013 19:04:47 -0500

Genome Browser is the platform/database used for searching and retreiving sequences and annotation of genomes belong to various eukaryotes, prokaryotes, etc.

Following are the weblink for different available browsers:

http://www.ensembl.org/index.html

http://ensemblgenomes.org/

http://genome.ucsc.edu/

http://www.ncbi.nlm.nih.gov/genome

http://www.ebi.ac.uk/genomes/

http://flybase.org/

http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi

http://www.sanger.ac.uk/resources/databases/