Address of the bookmark: http://readiab.org/

More at https://pubmed.ncbi.nlm.nih.gov/20494974/

Address of the bookmark: http://www.sequenceontology.org/cgi-bin/soba.cgi

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

1. Batch Effects: Technical variability can confound biological signals. Combat this with normalization techniques or batch-correction tools like ComBat.
2. Low-Quality Samples: Poor-quality RNA impacts downstream analyses.
3. 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.

List of analysis tools developed for Oxford Nanopore data

BWA
Fast nanopore data tuned alignment tool
https://github.com/lh3/bwa

GraphMap
Mapper for long and error-prone reads
https://github.com/isovic/graphmap

LAST
Nanopore tuned alignment tool
http://last.cbrc.jp/

LINKS
Software tool for long read scaffolding
https://github.com/warrenlr/LINKS/

marginAlign
Tools to align nanopore reads to a reference
https://github.com/benedictpaten/marginAlign

minoTour
Real time analysis tools
http://minotour.nottingham.ac.uk/

nanoCORR
Error-correction tool for nanopore sequence data
https://github.com/jgurtowski/nanocorr

NanoOK
Software for nanopore data, quality and error profiles
https://documentation.tgac.ac.uk/display/NANOOK/NanoOK

Nanopolish
Nanopore analysis and genome assembly software
https://github.com/jts/nanopolish

nanopore
Variant-detection tool for nanopore sequence data
https://github.com/mitenjain/nanopore

Nanocorrect
Error-correction tool for nanopore sequence data
https://github.com/jts/nanocorrect/

npReader
Real-time conversion and analysis of nanopore reads
https://github.com/mdcao/npReader

poRe
Tool for analyzing and visualizing nanopore data
https://sourceforge.net/p/rpore/wiki/Home/

PoreSeq
Error-correction and variant-calling software
https://github.com/tszalay/poreseq

Poretools
Nanopore sequence analysis and visualization software
https://github.com/arq5x/poretools

SSPACE-LongRead
Genome scaffolding tool
http://www.baseclear.com/genomics/bioinformatics/basetools/SSPACE-longread

SMIS
Genome scaffolding tool
https://sourceforge.net/projects/phusion2/files/smis/

List of assemblers for Oxford Nanopore MinION long reads

LQS
DALIGNER, Celera OLC Nanocorrect,
Nanopolish corrector
https://github.com/jts/nanopolish

PBcR
HGAP or BLASR, Celera OLC
PBcR corrector
http://wgs-assembler.sourceforge.net/wiki/index.php/PBcR
–
Canu
MHAP, Celera OLC
Canu corrector
https://github.com/marbl/canu

Falcon
String graph, Celera OLC
Falcon corrector
https://github.com/PacificBiosciences/falcon

Miniasm
OLC
https://github.com/lh3/miniasm

ra-integrate
OLC
https://github.com/mariokostelac/ra-integrate/

ALLPATHS-LG
de Bruijn graph
ALLPATHS-L corrector
https://www.broadinstitute.org/software/allpaths-lg/blog/?page_id=12

SPAdes
de Bruijn graph
SPAdes corrector
http://bioinf.spbau.ru/spades

https://function.princeton.edu/

More at https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-020-03666-4

Address of the bookmark: https://github.com/aghozlane/shaman

University of Ibadan (UIB)­Ibadan:
There has been significant increase in the number of bioinformatics activities in Nigeria (and West Africa) since 2003 when the program was initiated by the West African Biotechnology Workshops Series (WABWS, http://www.wabw.org) at the University of Ibadan, Nigeria (in collaboration with  the South African National Bioinformatics Institute (SANBI, http:/www.sanbi.ac.za). Workshops  that were open to scientists from all African countries have seen a very high number of applications  from scientists based in West Africa. The encouraging desire to acquire cutting­edge skills to  computational process data and extract useful knowledge from genome projects led to the interest of  the West African Biotechnology Workshops (WABW) to develop an agenda to address the  bioinformatics skills gap among scientists in West Africa. An increased commitment from agencies  like NEPAD would be required in the provision of infrastructure to establish and sustain regional  and national networks.

University of Ilorin (UIL)­Ilorin:
The University of Ilorin was established in 1976 by the Federal Government of Nigeria.  Bioinformatics activities started at the University in February 2003 with the establishment of the  West African Bioinformatics Research Initiative (WABRI). However, progress has been rather slow  due to inadequate funding. We are mainly engaged in Bioinformatics training at the introductory  level and proteomics studies on various species of malaria parasites. Recently, we became interested  in comparative genome analysis of various species of  Plasmodium  and the comparison of  chloroquine sensitive and chloroquine resistant strains of Plasmodium falciparum. Other activities  and areas of interest can be seen on our website, http://www.wabri.org, although not all our  proposed interests have been fully implemented due to our level of funding.

Training:
The University of Ilorin has introduced M.Sc. and Ph.D. programmes in Computer Science (with  options in Bioinformatics). The programme is based in the Department of Computer Science and  emphasis is on the development of algorithms to solve problems in bioinformatics. The Covenant University offers M.Sc. and Ph.D in Computer Science with option in Bioinformatics  (Computational Biology). Furthermore, through affiliated departments, the CBBM is been design to award Diploma and Degree certificates in Biotechnology.

Web sites and links: http://www.covenantuniversity.com http://www.run.edu.ng http://www.uniben.edu http://www.wabri.org http://www.wabw.org http://www.unilorin.edu.ng http://www.wabri.org http://www.asopah.org

Software; Creation of Public Domain Resources in Biology
Present Status::

Developing prediction methods for gene, beta-turn, secondary structure and MHC-binding sites.
Area of Interest ::

Comparison of force field simulations. Analysis of DNA-protein interactions using molecular mechanics methods.Drug Target Identification using in silico biology.

More @ http://www.imtech.res.in/bic/index.php?option=com_content&view=article&id=65

PIs: http://www.imtech.res.in/bic/index.php?option=com_content&view=article&id=69

awk '/pattern1/ {Actions}
/pattern2/ {Actions}' file

The working of Awk is as follows
Awk reads the input files one line at a time.
For each line, it matches with given pattern in the given order, if matches performs the corresponding action.
If no pattern matches, no action will be performed.
In the above syntax, either search pattern or action are optional, But not both.
If the search pattern is not given, then Awk performs the given actions for each line of the input.
If the action is not given, print all that lines that matches with the given patterns which is the default action.
Empty braces with out any action does nothing. It wont perform default printing operation.
Each statement in Actions should be delimited by semicolon.
Say you have data.tsv with the following contents:

$ cat data/test.tsv
contig1 ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2 ACTTTATATATT
contig3 ACTTATATATATATA
contig4 ACTTATATATATATA
contig5 ACTTTATATATT
By default Awk prints every line from the file.

$ awk '{print;}' data/test.tsv
contig1 ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2 ACTTTATATATT
contig3 ACTTATATATATATA
contig4 ACTTATATATATATA
contig5 ACTTTATATATT
We print the line which matches the pattern contig3

$ awk '/contig3/' data/test.tsv
contig3 ACTTATATATATATA
Awk has number of builtin variables. For each record i.e line, it splits the record delimited by whitespace character by default and stores it in the $n variables. If the line has 5 words, it will be stored in $1, $2, $3, $4 and $5. $0 represents the whole line. NF is a builtin variable which represents the total number of fields in a record.

$ awk '{print $1","$2;}' data/test.tsv
contig1,ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2,ACTTTATATATT
contig3,ACTTATATATATATA
contig4,ACTTATATATATATA
contig5,ACTTTATATATT

$ awk '{print $1","$NF;}' data/test.tsv
contig1,ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2,ACTTTATATATT
contig3,ACTTATATATATATA
contig4,ACTTATATATATATA
contig5,ACTTTATATATT

Awk has two important patterns which are specified by the keyword called BEGIN and END. The syntax is as follows:

BEGIN { Actions before reading the file}
{Actions for everyline in the file}
END { Actions after reading the file }

For example,
$ awk 'BEGIN{print "Header,Sequence"}{print $1","$2;}END{print "-------"}' data/test.tsv
Header,Sequence
contig1,ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2,ACTTTATATATT
contig3,ACTTATATATATATA
contig4,ACTTATATATATATA
contig5,ACTTTATATATT
-------
We can also use the concept of a conditional operator in print statement of the form print CONDITION ? PRINT_IF_TRUE_TEXT : PRINT_IF_FALSE_TEXT. For example, in the code below, we identify sequences with lengths > 14:

$ awk '{print (length($2)>14) ? $0">14" : $0"<=14";}' data/test.tsv
contig1 ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG>14
contig2 ACTTTATATATT<=14
contig3 ACTTATATATATATA>14
contig4 ACTTATATATATATA>14
contig5 ACTTTATATATT<=14
We can also use 1 after the last block {} to print everything (1 is a shorthand notation for {print $0} which becomes {print} as without any argument print will print $0 by default), and within this block, we can change $0, for example to assign the first field to $0 for third line (NR==3), we can use:

$ awk 'NR==3{$0=$1}1' data/test.tsv
contig1 ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2 ACTTTATATATT
contig3
contig4 ACTTATATATATATA
contig5 ACTTTATATATT
You can have as many blocks as you want and they will be executed on each line in the order they appear, for example, if we want to print $1 three times (here we are using printf instead of print as the former doesn't put end-of-line character),

$ awk '{printf $1"\t"}{printf $1"\t"}{print $1}' data/test.tsv
contig1 contig1 contig1
contig2 contig2 contig2
contig3 contig3 contig3
contig4 contig4 contig4
contig5 contig5 contig5
Although, we can also skip executing later blocks for a given line by using next keyword:

$ awk '{printf $1"\t"}NR==3{print "";next}{print $1}' data/test.tsv
contig1 contig1
contig2 contig2
contig3
contig4 contig4
contig5 contig5

$ awk 'NR==3{print "";next}{printf $1"\t"}{print $1}' data/test.tsv
contig1 contig1
contig2 contig2

contig4 contig4
contig5 contig5
You can also use getline to load the contents of another file in addition to the one you are reading, for example, in the statement given below, the while loop will load each line from test.tsv into k until no more lines are to be read:

$ awk 'BEGIN{while((getline k <"data/test.tsv")>0) print "BEGIN:"k}{print}' data/test.tsv
BEGIN:contig1 ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
BEGIN:contig2 ACTTTATATATT
BEGIN:contig3 ACTTATATATATATA
BEGIN:contig4 ACTTATATATATATA
BEGIN:contig5 ACTTTATATATT
contig1 ACTGTCTGTCACTGTGTTGTGATGTTGTGTGTG
contig2 ACTTTATATATT
contig3 ACTTATATATATATA
contig4 ACTTATATATATATA
contig5 ACTTTATATATT
You can also store data in the memory with the syntax VARIABLE_NAME[KEY]=VALUE which you can later use through for (INDEX in VARIABLE_NAME) command:

$ awk '{i[$1]=1}END{for (j in i) print j"<="i[j]}' data/test.tsv
contig1<=1
contig2<=1
contig3<=1
contig4<=1
contig5<=1

https://flowingdata.com/

Address of the bookmark: https://flowingdata.com/