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Linux Commands

This document provides instructions for performing various data analysis tasks in Linux/Unix including: 1. Installing necessary tools like Bowtie2, Velvet, Samtools, and VarScan for alignment, assembly, variant calling, and other tasks. 2. Using these tools to align reads with Bowtie2, assemble transcripts with Velvet, convert formats and sort BAM files with Samtools, and call SNPs and indels with VarScan. 3. It also describes how to use tools like BAMView to view alignments.

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59 views3 pages

Linux Commands

This document provides instructions for performing various data analysis tasks in Linux/Unix including: 1. Installing necessary tools like Bowtie2, Velvet, Samtools, and VarScan for alignment, assembly, variant calling, and other tasks. 2. Using these tools to align reads with Bowtie2, assemble transcripts with Velvet, convert formats and sort BAM files with Samtools, and call SNPs and indels with VarScan. 3. It also describes how to use tools like BAMView to view alignments.

Uploaded by

aleena.faizyab
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Data Analysis in Linux/Unix

#For window10 or above, go to start menu, SEARCH UBUNTU ON MICROSOFT STORE,


click on download/install Ubuntu (version 22.04).
# After installing Ubuntu (version 22.04), open the app, provide a name and password
(remember that, or store it at some safe place for future use).
# After doing the above, update/upgrade the system by entering following commands on
linux terminal.
sudo apt update
sudo apt upgrade
# means comments, rest are all commands
pwd #present working directory (the directory you’re in)
ls #lists files and directories
ls –a #shows hidden files
ls –lh #shows details, such as file sizes
cd Desktop #changes current directory to Desktop
cd .. #Changes to one directory “higher”
cd / #changes to “root” directory
cd ~ #go to home directory
mkdir workshop #makes a new directory named workshop
rm workshop # removes directory if empty else use rm –r
nano text #creates, views/edits contents of file “text”
more text #see file “text” in screen-size chunks
less text #see file with arrows to move about (type :q to quit)
head text #see top of file “text”
tail text #see bottom of file “text”
cp text text1 #will copy contents of file text and save it as text1
cp text text #Try it, will curse you 
cp text test #creates copy of the file “text” in the dir test
#If there is no directory called test, creates the file test with contents as of file test, If there is
already a file called test: it will be automatically over-written!
mv text test #Moves contents, except it deletes the original file
rm text #Deletes the file “text”. Forever.
gunzip xxx.gz #unzip a .gz compressed file
unzip xxx.zip #unzip a .zip compressed file
tar –xvf #un-tar a tar archive
man tar # will open tar manual and helps about program “tar”
top #see all system processes
ssh #open a remote, secure connection
ctrl-z #stop a program
kill %1 #get rid of it for good
grep this file.txt #finds lines matching “this” in file text
ftp #get a remote file
#How to run scripts/codes written in other languages like perl/python etc in linux
python myscript.py # runs a python script.
NGS Data Analysis in Linux
# We need following Linux/Unix based tools for our NGS data analysis, we can get them one
by one as;
# 1: bowtie2 is a short read aligner. Used for Alignment of reads to transcripts/Gene
Models or Genome, important in RNASeq and Variant calling
sudo apt-get install bowtie2 # sudo will ask for password etc
# Running bowtie2
bowtie2-build AT_Transcripts.txt AT_Indexes # mac users will add ./ in the beginning
(./bowtie-)
# will build indexes from the transcripts/Gene Models, that will help aligning of the reads
quickly and efficiently
bowtie2 -x AT_Indexes mu1.txt > mu1.output # mac users (./bowtie2 -x AT -----)
# Will align the reads stored in file mu1.txt to the indexes we made in the above
python3 getCounts_updated.py -in mu1.output
# Counting the reads, aligned to various gene models
# 2 Genome/Transcriptome Assembly. The tools are called assemblers, we will use one
of the known as velvet
sudo apt install velvet
velveth 31mer 31 -fastq -short mu1.txt #mac users (./velveth----)
#velveth is algorithm for hash/kmer generation in velvet program.
#foldername can be any output folder name in which you store data. If this name is not given
file will get stored in present directory.
#31 is kmer length. Velvet comes with by-default maximum 31kmer length. This can be
changed during installation.
#For velvet you need to tell the program file format & type of input file. So that it can
distinguish between different file formats.
velvetg 31mer/ #mac users (./velvetg ---)
#velvetg is algorithm for making contigs in velvet program.
#min_contig_lgth this should be greater than kmer length. So that contigs can be made using
overlaps.
# 3 Variant Calling. We will align the files with aligners like bowtie, bwa or others and
use the output file in sam format
sudo apt install samtools
samtools view -S -b mu1.output > mu1_output.bam
# to convert sam file to bam, mac users don’t forget to add ./
samtools sort mu1_output.bam -o mu1_output.bam.sorted
# will sort the bam file (./ for mac)
samtools mpileup -f AT_Transcripts.txt mu1_output.bam.sorted > mu1_mplieup_results
# (./ mac)
#Call SNPs using varscan
For this we will use chromosome 22 data, to have significant snps and indels
bowtie2-build chr22.fasta chr22_indexes
bowtie2 -x chr22_indexes chr22.fastq > chr22.output
samtools view -S -b chr22.output > chr22.output.bam
samtools sort chr22.output.bam -o chr22.output.bam.sorted
samtools mpileup -f chr22.fasta chr22.output.bam.sorted > chr22.mplieup_results
We need java to be installed on computers
Java # will return you help if java is installed, else error message will be displayed
sudo apt install default-jre # will install Java on ubuntu
java -jar VarScan.v2.3.9.jar pileup2snp chr22.mplieup_results > chr22.snp.txt
# SNP calling
Call indels with varscan
java -jar VarScan.v2.3.9.jar pileup2indel chr22.mpileup > chr22.idels.txt # INDELS calling
###############################
Alignment viewer: BAMVIEW
Setup and installation Bamview:
java -mx512m -jar BamVi_v1.2.11.jar

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