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Bioinformatics for biologists
Dr. Habil Zare, PhDPI of Oncinfo Lab
Assistant Professor, Department of Computer Science Texas State University
Presented at University of Texas, Health Science Center – San Antonio20 November 2015
Part 1
- BioLinux - Mapping RNAseq data to transcriptome (Salmon)
Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 2015
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Bioinformatics: Computational and statistical analysis of biological data
Data
Biologists
ResultsGenotypes / Phenotypes
Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 2015
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In this workshop: A compact demo of bioinformatics analysis starting from raw data to produce useful plots and meaningful interpretation of the data
RNAseq
Biologists
Pathway and Network Analysis
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Goals of the workshop
- A practical introduction to some basic bioinformatics tools for biologists.
- Having hands-on experience with simple, toy-example data.
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Bio-Linux
Bio-Linux is a free workstation platform that facilitates running hundreds of bioinformatics tools without the corresponding installation hassles.
An easy way to install it on Mac OS X and Windows computers is described below:http://oncinfo.org/file/view/BioLinux_VM.pptx/564155065/BioLinux_VM.pptx
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Browsing files and folders
tar.gz refers to a compressed file in Linux. Let’s practice decompressing such a file with an example. Follow the next steps in BioLinux.
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.Double-click on Bio-Linux
Documentation to open it.
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.
Double-click on Introductory Tutorial
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.Click on File>New TAb
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.Select the second tab and click
on Home.
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.
Drag and drop this file from intro_course tab to Home tab.
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.
Right-click on the file and then Extract Here…
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.This folder will appear. Open it
and have a look inside.
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.
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Downloading and installing
Most useful bioinformatics tools are publicly available. You can download, install, and use them easily.
Let’s practice with an example. Follow the next steps in BioLinux.
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.
This is the “Dash”. Use it to launch and organize applications.
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.E.g., use “Firefox” to browse the web.
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.Type oncinfo.org in the address bar and press enter.
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From the right menu, click on the workshop link.
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.
Click on “zipped” to download the folder.
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.
Choose to save the file.
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1- Click on Files icon.
2- Click on Downloads.
The file that you just downloaded was saved in Downloades folder.
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This is the file you just downloaded.
The file that you just downloaded was saved in Downloades folder.
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Extract (decompress) the file that you just .
Right-click on the file and then Extract Here…
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The file that you just downloaded in saved in Downloades folder.
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Salmon
Salmon, a successor of Sailfish, is a useful tool for mapping RNAseq data. It is faster and easier to run than alternatives such as TopHat.
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Installing Salmon software
We will run a script provided in the zipped file using a terminal.
Terminal is an interface that uses only text to communicate between the user and the computer.
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.
Click on the black rectangular to open a terminal.
How to open a terminal?
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.Try a few simple Linux commands e.g.,echo, date, cal, …
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.
Type “cd” in the terminal to “change directory”.
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.
Drag the folder to the terminal.
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.
Now press Enter.
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. Double-click on the folder to open it.
What is in the folder?
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Equivalently, “ls” shows you the list
of files in this folder.
What is in the folder?
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This script will install Salmon for you.
What is in the folder?
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Type the name of the script
and then press Enter.
How to run the script?
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How to run the script?
Type your password, which is “manager” by default.
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How to make sure Salmon is installed?
Type “salmon v” to test if it is installed or not.
The script should download and install salmon. The following test indicates that installation was OK.
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1- A FASTA file, which has the sequence information of the transcriptome of the species of interest.
2- One or more FASTQ files, which are provided by the sequencer instrument and contain the reads information from the samples.
Input for Salmon
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Toy examples of FASTA and FASTQ files
Open the sample_data folder
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Next generation sequencing A sequencer produces millions of short reads (50-200 bps).
Biological sample Sequencer Short reads
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Toy examples of a FASTQ file
Double click on reads_1.fastq file.
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This is a read of length 50 with nucleotide and (Phred) quality information.
Toy examples of a FASTQ file
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Double click on transcripts.fasta file.
Toy examples of a FASTA file
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This is a transcript.
Toy examples of a FASTA file
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It is an mRNA with RefSeq ID NM_001168316
Toy examples of a FASTA file
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Type the RefSeq ID, e.g., NM_001168316
More information on the transcript Search in the NCBI database http://www.ncbi.nlm.nih.gov/nuccore/
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Type the RefSeq ID, e.g., NM_001168316
Visualize the transcript on the genome Search in the UCSC genome browserhttps://genome.ucsc.edu/cgi-bin/hgGateway
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This is the transcript
Visualize the transcript on the genome Search in the UCSC genome browserhttps://genome.ucsc.edu/cgi-bin/hgGateway
Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 2015
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More information on this region is available.
Visualize the transcript on the genome Search in the UCSC Genome Browserhttps://genome.ucsc.edu/cgi-bin/hgGateway
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Quantify the level of expressionThe level of expression of each transcript can be quantified by counting the number of reads that are aligned to it.
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Next generation sequencing A sequencer produces millions of short reads (50-200 bps).
Biological sample Sequencer Short reads
Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 2015
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Only exons are present in mRNA
} } } }
exon 1 exon 2 exon 3 exon 4
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Alignment
Gene 1 Gene 2
Determines what transcript (where on the genome) each read was originated from.
Short reads in a FASTQ file
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Alignment
Gene 1 Gene 2
Short reads in a FASTQ file
Determines what transcript (where on the genome) each read was originated from.
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Alignment
Gene 1 Gene 2
Count the number of aligned (mapped) reads to each region.
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Alignment
Gene 1 Gene 2
High expression Low expression
Compare the level of expression between genes.
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Quantifying expression from RNAseq data
Salmon processes raw data and quantifies expression levels in 2 steps.http://salmon.readthedocs.org/en/latest/salmon.html#using-salmon
Step 1- Building an index for the transcriptome. Step 2- Aligning the reads to the transcriptome.
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Are you in the right directory?Before you start, make sure you are in the correct directory.The pwd command in Linux shows the current directory.
Typing “pwd” and then “Enter” will “print the working directory”, i.e., your current path.
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Always make sure that the files are stored where you expect them to be.
Are you in the right directory?Before you start, make sure you are in the correct directory.The pwd command in Linux shows the current directory.
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Step 1- Building an index for the transcriptome.
Run the following command in the terminal in BioLinux:
salmon index -t transcripts.fasta -i transcripts_index --type fmd
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Type the command here.
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For now, ig
nore this
warning.
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The index is built.
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Salmon created a new folder.
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Step 2- Aligning the reads to the transcriptome.
Run the following command in the terminal in BioLinux:
salmon quant -i transcripts_index –l IU -1 reads_1.fastq -2 reads_2.fastq –o transcripts_quanton
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Step 2- Aligning the reads to the transcriptome.
Run the following command in the terminal in BioLinux:
}
The command
}
The indexing built in step 1
}
The first input file
}The secondinput file
}
Output folder
salmon quant -i transcripts_index –l IU -1 reads_1.fastq -2 reads_2.fastq –o transcripts_quanton
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Salmon created a new folder and stored the results there.
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quant.sf is the main output file that reports the number of reads and expression. Double click on it.
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The names of the transcripts (RefSeq IDs) and their length are in the first 2 columns.
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The number of mapped reads is reported on the last column.
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Transcript per million (TPM) is the estimated expression.
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Transcript per million (TPM) is the estimated expression.
TPM values correspond to counts normalized by the length of transcripts and also the depth of sequencing. There are other normalization methods such as RPKM and FPKM.
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This transcript is highly expressed
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This transcript is highly expressed
These transcripts have low expression.
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References:
• Some of the slides are based on Introduction to Biolinux http://nebc.nerc.ac.uk/downloads/courses/Bio-Linux/bl8_latest.pdf
• Salmon is a useful tool for mapping and analyzing RNAseq data. https://combine-lab.github.io/salmon/
• I prepared these guidelines to facilitate the “Bioinformatics for biologists workshop”, 20 Nov 2015, UTHSC – San Antonio.http://oncinfo.org/Bioinformatics+for+biologist+workshop