Bioinformatics for biologists Dr. Habil Zare, PhD PI of Oncinfo Lab Assistant Professor, Department of Computer Science Texas State University Presented at University of Texas, Health Science Center – San Antonio 20 November 2015

Part 1 - BioLinux - Mapping RNAseq data to transcriptome (Salmon)

Bioinformatics: Computational and statistical analysis of biological data Data Biologists Results Genotypes / Phenotypes Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

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 Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

Goals of the workshop -A practical introduction to some basic bioinformatics tools for biologists. -Having hands-on experience with simple, toy-example data. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

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: Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

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. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

. 8 Double-click on Bio-Linux Documentation to open it.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Double-click on Introductory Tutorial

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Click on File>New TAb Click on File>New TAb

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Select the second tab and click on Home.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Drag and drop this file from intro_course tab to Home tab.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Right-click on the file and then Extract Here…

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This folder will appear. Open it and have a look inside.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

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. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This is the “Dash”. Use it to launch and organize applications.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov E.g., use “Firefox” to browse the web.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Type oncinfo.org in the address bar and press enter.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov From the right menu, click on the workshop link.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Click on “zipped” to download the folder.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Choose to save the file.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Click on Files icon. 2- Click on Downloads. The file that you just downloaded was saved in Downloades folder.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This is the file you just downloaded. The file that you just downloaded was saved in Downloades folder.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Extract (decompress) the file that you just. Right-click on the file and then Extract Here…

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov The file that you just downloaded in saved in Downloades folder.

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. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

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. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Click on the black rectangular to open a terminal. Click on the black rectangular to open a terminal. How to open a terminal?

. Try a few simple Linux commands e.g., echo, date, cal, … Try a few simple Linux commands e.g., echo, date, cal, … Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Type “ cd ” in the terminal to “ c hange d irectory”.

. Drag the folder to the terminal. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Now press Enter.

. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Double-click on the folder to open it. What is in the folder?

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Equivalently, “ls” shows you the list of files in this folder. What is in the folder?

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This script will install Salmon for you. What is in the folder?

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Type the name of the script and then press Enter. Type the name of the script and then press Enter. How to run the script?

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov How to run the script? Type your password, which is “manager” by default.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 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.

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. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Input for Salmon

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Toy examples of FASTA and FASTQ files Open the sample_data folder

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Next generation sequencing A sequencer produces millions of short reads ( bps). Biological sample Sequencer Short reads

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Toy examples of a FASTQ file Double click on reads_1.fastq file.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This is a read of length 50 with nucleotide and (Phred) quality information. Toy examples of a FASTQ file

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Double click on transcripts.fasta file. Toy examples of a FASTA file

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This is a transcript. Toy examples of a FASTA file

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov It is an mRNA with RefSeq ID NM_ Toy examples of a FASTA file

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Type the RefSeq ID, e.g., NM_ More information on the transcript Search in the NCBI database

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Type the RefSeq ID, e.g., NM_ Visualize the transcript on the genome Search in the UCSC genome browser bin/hgGateway bin/hgGateway

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This is the transcript Visualize the transcript on the genome Search in the UCSC genome browser bin/hgGateway bin/hgGateway

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov More information on this region is available. Visualize the transcript on the genome Search in the UCSC Genome Browser bin/hgGateway bin/hgGateway

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Quantify the level of expression The level of expression of each transcript can be quantified by counting the number of reads that are aligned to it.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Next generation sequencing A sequencer produces millions of short reads ( bps). Biological sample Sequencer Short reads

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Only exons are present in mRNA } } } } exon 1 exon 2 exon 3 exon 4

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Alignment Gene 1Gene 2 Determines what transcript (where on the genome) each read was originated from. Short reads in a FASTQ file

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Alignment Gene 1Gene 2 Short reads in a FASTQ file Determines what transcript (where on the genome) each read was originated from.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Alignment Gene 1Gene 2 Count the number of aligned (mapped) reads to each region.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Alignment Gene 1Gene 2 High expressionLow expression Compare the level of expression between genes.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Quantifying expression from RNAseq data Salmon processes raw data and quantifies expression levels in 2 steps. Step 1- Building an index for the transcriptome. Step 2- Aligning the reads to the transcriptome.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 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.

Always make sure that the files are stored where you expect them to be. Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 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.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 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

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Type the command here.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov For now, ignore this warning.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov The index is built.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Salmon created a new folder.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 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

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov 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 second input file } Output folder salmon quant -i transcripts_index –l IU -1 reads_1.fastq -2 reads_2.fastq –o transcripts_quanton

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Salmon created a new folder and stored the results there.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov quant.sf is the main output file that reports the number of reads and expression. Double click on it.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov The names of the transcripts (RefSeq IDs) and their length are in the first 2 columns.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov The number of mapped reads is reported on the last column.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Transcript per million (TPM) is the estimated expression. Transcript per million (TPM) is the estimated expression.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov Transcript per million (TPM) is the estimated expression. 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.

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This transcript is highly expressed

Bioinformatics for biologists workshop, Dr. Habil Zare, Oncinfo Lab UTHSC San Antonio, 20 Nov This transcript is highly expressed These transcripts have low expression.

References: Some of the slides are based on Introduction to Biolinux Salmon is a useful tool for mapping and analyzing RNAseq data. I prepared these guidelines to facilitate the “Bioinformatics for biologists workshop”, 20 Nov 2015, UTHSC – San Antonio. Instaling BioLinux using VM, Dr. Habil Zare 27 Oct