Unix commands for beginners

D. Puthier TAGC/Inserm, U1090, [email protected] Matthieu Defrance, ULB, matthieu.dc[email protected] Stéphanie Le gras, Igbmc, [email protected] Christophe Blanchet, IFB, [email protected] MATE Desktop

Demo Quick overview.

Installation: http://www.france-bioinformatique.fr/?q=fr/core/cellule-infrastructure/documentation-cloud

Dashboard: https://cloud.france-bioinformatique.fr/cloud/instance/ The terminal…

Demo Type ‘ls’ in the terminal (list files)

# list files root@vm: ls How can I speak to the terminal

● Answer : you can speak in BASH (Bourne Again Shell) * ○ BASH is one of numerous shell dialect (ksh, csh, zsh,...). ○ All this shell languages are extremely similar. ○ These languages are based on commands. ○ These modular commands allows one to perform tasks.

* Reférence (calembour) au premier langage Shell écrit par Stephen Bourne :) Command prototype(s) (1)

● One command performs a task (sort, select, open, align reads,...). ● A command has arguments that may be facultative and modify the way it works. ● These arguments may take some values. ● Most of the time an instruction (command line) starts with a command name (or path to the command). ● In the example below we will say minus v’.

# Argument without any associated value # depending on the command v means verbose, version (or other) fastqc -v

# An argument with an associated value man -k jpeg Command prototype(s) (1)

● Most of the time arguments can be written in their short of long form (more explicit/better readability). ● Long form are generally precede with ‘--’ (for instance ‘minus minus apropos’)

# Long form without any associated value. fastqc --version

# Long form with an associated value. man --apropos jpeg Getting help !

Call you friends or better use man (manuel)

# Demo root@vm: man ls # getting help about ls root@vm: man man # getting help about man ...

Help shortcuts: /foo : search for ‘foo’. n : (next) next occurence of ‘foo’. p: (previous) previous occurrence of ‘foo’. q : quit help page. Our first command: ls The ls command and some of its arguments

● ls can take several arguments. ● Main arguments: ○ -l : (long) get lot of information. ○ -a (all) show all files including hidden files*. ○ -1 : show results as 1 column. ○ -t (time) sort results by date/time. ○ -r (reverse) reverse sort order. ● One can combine arguments ○ ls -l -a ○ ls -la

* Under linux hidden files start with a ‘.’ (e.g ‘.thehiddenfile.txt’). The ls command and some of its arguments

# Demo root@vm: ls # list files root@vm: ls -a # list files including hidden files * root@vm: ls -l # get lot of information about files root@vm: ls -1 # list file (one column) root@vm: ls -t # List file by modification date **

# Combining arguments root@vm: ls -rtl # lot of info, sort by date, reverse order

* WARNING with spaces. Instruction should start with a command. The ls-a command does not exists !

** Default sorting is case-sensitive sorting. Create directories and files File system tree

● The file system can be viewed as a tree in which nodes are directories or files. ● This tree has a root: /

● The root folder (/) contains ○ A root folder an various additional folder* ■ Under IFB machine your root folder contains a Documents folder

* Under IFB VM, you are the root/sysadmin, this is a particular case. Hos should I refer to a file/directory

● 1) By specifying the path from the root. Absolute path.

e.g; /root/Documents /root/Music

● 2) By referring to the current location/directory (the working/current directory). Relative path. Syntax for relative path

# The upper directory relative to the working directory ..

# Two directories up ../.. # Three ../../..

# The current working directory

./ File system: Demo

pwd (print working directory); cd (change directory). *

root@vm: pwd # The current working directory (/root) root@vm: cd /root/Documents # We go into Documents root@vm: pwd # /root/Documents root@vm: cd .. # go up one level (/root) root@vm: cd /root/Music # Go to the Music folder root@vm: pwd # /root/Music root@vm: cd ../.. # Go to the root of the file system root@vm: ls # You should see the root directory root@vm: cd /root/Music # Let’s go to the root/Music directory root@vm: cd ../Documents # And to the Document folder

* Use complétion (tab key) for file, directories and commands. File system: some hints

● If you are the /root your data are stored in /root ○ i.e ‘user directory’ or home. ● ~ (tilda) contains the path to your home (same as $HOME). root@vm: cd / # At the root root@vm: pwd # / root@vm: cd ~/Documents # The Document directory of your home folder. root@vm: cd ~ # go to your home dir. root@vm: cd /usr/local/bin # Go to /usr/local/bin root@vm: ls ~ # list files in your ‘home’ directory root@vm: cd ~/Music # Go to the Music folder inside your home dir. root@vm: cd # == cd ~ Make directories

● We will use the mkdir (make directory) command.

root@vm: mkdir projet_roscoff # Create a directory root@vm: cd ./projet_roscoff # == cd projet_roscoff * root@vm: mkdir rna-seq # Let’s create a folder root@vm: mkdir chip-seq dna-seq # and several sub-folders root@vm: ls -1 # list files and folders root@vm: cd chip-seq # == ./chip-seq root@vm: pwd # the current working dir root@vm: cd ../.. # go back home

* ./ is most of the time facultative Hands on

● 1) go to ~/projet_roscoff/chip-seq ● 2) From this directory create a directory named annotation in ~/projet_roscoff/ ● Go inside annotation directory ● Check you are in the write place ● Go back home. Hands on

● 1) go to ~/projet_roscoff/chip-seq ● 2) From this directory create a directory named annotation in ~/projet_roscoff/ ● Go inside annotation directory ● Check you are in the write place ● Go back home. ● # Solution root@vm: cd ~/projet_roscoff/chip-seq root@vm: mkdir ../annotations root@vm: cd ../annotations root@vm: cd Manipulate files Download and uncompress files

● We will use the wget command to download files. ● To uncompress we will use gunzip if the file was compressed with the gzip algorithm (extension .gz) root@vm: cd ~/projet_roscoff/annotations # on se déplace dans annotations

# On télécharge le fichier root@vm: wget http://pedagogix-tagc.univ-mrs.fr/courses/data/roscoff/hg19_exons.bed.gz root@vm: ls # le fichier compressé root@vm: ls # le fichier compressé root@vm: gunzip hg19_exons.bed # on le décompresse root@vm: ls # le fichier a perdu l’extension gz The hg19_exons.bed file

Contains coordinates (start/end) of humand exons in bed format.

Bed format (Bed6) ( http://genome.ucsc.edu/FAQ/FAQformat.html#format1 ) *

Tabulated format (how to check that ???)

Chromosome Start End Name Score Strand (Others…)

* Start and End position are always given relative to the 5’/3’ orientation of the + strand. Coordinates are ‘zero-based, half-open’. Visualising file content ● With a pager: less or more (do more or less the same). ● With head ou tail to display the n first or n last lines of a file. ● The cat command allows to send file content to the screen. + c to cancel. ● The shortcuts for less are the same as for the man command.

Raccourcis dans less: ↑ : go up. ↓ : go down. > : go to first line. < : go to last line. /foo : search for ‘foo’. n : next occurrence of foo. p: previous occurrence of foo q : quit. Hands on

● 1) Look at the ten first lines of hg19_exons.bed with head. ● 2) look at the ten last lines of hg19_exons.bed with tail. ● 3) Go through the hg19_exons.bed file with less. ● 4)Send file content to the screen with cat. Exercices

● 1) Look at the ten first lines of hg19_exons.bed with head. ● 2) look at the ten last lines of hg19_exons.bed with tail. ● 3) Go through the hg19_exons.bed file with less. ● 4)Send file content to the screen with cat.

# Solution root@vm: head -n 10 hg19_exons.bed root@vm: tail -n 10 hg19_exons.bed root@vm: less hg19_exons.bed root@vm: cat hg19_exons.bed Counting line number

This can be done with the wc (word count) command with -l (line) argument. root@vm: wc -l hg19_exons.bed # 484127 exons

Extract columns

● Use the cut command with the -f (field) argument ● The columns must be tabulated or use the -d argument (‘delimiter’)

root@vm: cut -f1 hg19_exons.bed # Column 1 root@vm: cut -f1,2 hg19_exons.bed # Columns 1 and 2 root@vm: cut -f3-5 hg19_exons.bed # Columns from 3 to 5 root@vm: cut -f3- hg19_exons.bed # Column 3 to the last column Sort a file

● On should use the sort command (alphabetic sorting by default). ○ -k (key): e.g ■ -k1,1: sort by column 1. ■ -k2,2nr: sort by column 2 using a numeric sorting in reverse order. ■ -k2,2g: sort by column 2 (decimal sorting).

Example: sort hg19_exons.bed by chromosomes then by genomic coordinates:

root@vm: sort -k1,1 -k2,2nr hg19_exons.bed Redirections Command pipes

Input Output Input Output Input Output

Commande Commande Commande

Error Error Error

● Standard Input: a file or text stream. ● Standard output: screen by default. ● Standard error: may be capture for log purpose. Demo: command pipes

Obtenir la liste de chromosomes présents dans le fichier root@vm: cut -f1 hg19_exons.bed | sort | uniq # La liste non-redondante des chromosomes

Obtenir la liste des chromosomes présents dans le fichier et leur nombre root@vm: sort hg19_exons.bed | uniq -c # -c pour ‘count’

Compter le nombre de transcript non codant (contenant ‘NR_’). root@vm: cut -f4 hg19_exons.bed | grep "NR_" | sort | uniq | wc -l #11675

Note: La commande uniq permet d’éliminer les doublons dans un fichier trié. Note: la commande grep permet de chercher une chaîne de caractères. Exercices (notés)

● How many exons on chromosome 22 ? ● What is the most frequent chrom-start-end tuple ? ○ i.e The most frequent exon. Exercices (notés)

● How many exons on chromosome 22 ? ● What is the most frequent chrom-start-end tuple ? ○ i.e The most frequent exon.

Solution root@vm: grep -w chr22 hg19_exons.bed | wc -l # n = 259 root@vm: cut -f1-3 hg19_exons.bed | sort | uniq -c| sort -n| tail -n 1 # 77 chrY

Exercice

● What is the genome fraction covered by exons ? ○ We must perform the operation below

Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon

Exon Exon Exon Exon

● Let see how to do that... Using Bedtools Bedtools

● A software to perform arithmetic operations on genomic coordinates. ○ http://bedtools.readthedocs.org/en/latest/content/overview.html

● Some example usages: ○ Extend/slop regions. ○ Compare regions (intersect). ○ Merge regions. ○ Format convertion. ○ … ● The bedtools command is associated with a set of sub-commands. Exercice with bedtools

● Use bedtools with -h argument. ○ What do you see ?

● Ask for some help about the merge command (bedtools merge -h) ○ Looks at the arguments. ○ Read the note at the end of the command. Why is it important ? Exercice with bedtools

● Use bedtools with -h argument. ○ What do you see ?

● Ask for some help about the merge command (bedtools merge -h) ○ Looks at the arguments. ○ Read the note at the end of the command. Why is it important ? ● Solution root@vm: bedtools -h # l’ensemble des sous commandes root@vm: bedtools merge -h # utiliser l’argument -i # la note indique que les régions génomiques doivent être triées au préalable. Exercice

● Use bedtools sort and bedtools merge to merge overlapping regions/exons. Exercice

● Use bedtools sort and bedtools merge to merge overlapping regions/exons.

root@vm: bedtools sort -i hg19_exons.bed | bedtools merge How to save results to a file ?

● Use the > redirection operator. ○ Erase file if it exists. ● >> can be used to add lines to an existing file.

root@vm: bedtools sort -i hg19_exons.bed | bedtools merge > hg19_exons_merged.bed root@vm: ls # A new file was created Some arithmetic with awk Awk

● Awk is a command available on most linux system. ● Awk has its own language. ● Awk allows to perform oneliners (and more) ● The prototype of a awk command is the following:

awk ‘BEGIN{action} {action} END{action}’ fichier

● Each set of brace is associated to a particular task:

BEGIN{before opening the file}

{for each line}

END{after rading all lines} Awk

● Awk has special variables. ● Examples:

FS: Field Separator.

OFS: Output Field Separator.

NR: Number of Row.

NF: Number of Field.

$0: The current line

$1,$2,$3 (...): columns 1,2 ou 3 (...) of the current line Exemple

# print columns 2 and 1 # \t is the tabulation character root@vm: awk 'BEGIN{FS="\t"}{print $2,$1}' hg19_exons.bed

# print columns 2 and 1 with tabulated output root@vm: awk 'BEGIN{FS=OFS="\t"}{print $2,$1}' hg19_exons.bed

# print columns 2 and 1 with tabulated output and line number root@vm: awk 'BEGIN{FS=OFS="\t"}{print NR,$2,$1}' hg19_exons.bed

# Compute start - end for each line root@vm: awk 'BEGIN{FS=OFS="\t"}{print $3-$2}' hg19_exons.bed Exercice

Calculer la somme des fragments (awk) Exercice: Calculer la somme des fragments (awk)

# Calculer à chaque ligne la somme cumulée de la taille des fragments # Notez que les “;” permettent de séparer des instructions # s est une variable que l’on déclare à 0 # 75861726 root@vm: awk 'BEGIN{FS="\t"; s=0}{s=s+$3-$2; print s}' hg19_exons_merged.bed

# Ou encore awk 'BEGIN{FS="\t"; s=0}{s=s+$3-$2}END{print s}' hg19_exons_merged.bed

# A vos calculettes (vous pouvez utiliser R). # 75861726/3.2e9*100 # ~ 2.37 % du génome couvert Aller plus loin avec awk

● Le prototype d’une commande awk peut être un peu étendu en ajoutant des ‘patterns’ (selecteurs ou critères).

awk ‘BEGIN{} pattern {} END{}’ fichier

● Le critère pourra être une expression régulière (voir plus loin) ou une expression logique

# exemples: test si a égal b. Imprime si vrai.

awk ‘a == b {} END{}’ fichier

# Exemples: imprime si la colonne 1 vérifie une expression régulière.

awk ‘$1 ~/regExp/ {print}’ fichier Exemples avec des patterns

# La première ligne root@vm: awk 'NR == 1 {print}' hg19_exons_merged.bed

# La ligne 2 à 10 root@vm: awk '{OFS=”\t”} NR >= 2 && NR <= 10 {print NR, $0}' hg19_exons_merged.bed

# Les lignes dont la colonne 1 contient la chaîne ‘chr19’. root@vm: awk ' $1 ~/chr19/ {print}' hg19_exons_merged.bed

Expressions régulières

● Permettent de décrire un motif dans une chaîne de caractère.

. un caractère quelconque

[a-z] une lettre minuscule (interval, ex : [u − w])

[A-Z] une lettre majuscule (interval, ex : [U − W])

[ABc] A ou B ou c

[ˆABab] Toute lettre différente de a et b.

^ Début de ligne.

$ Fin de ligne

x* 0 à n fois le caractères x.

x+ 1 à n fois le caractères x.

x{n,m} Le caractère x répété n à m fois. Exemples

\.txt$ Toute chaîne finissant par “.txt”

ˆ[A − B] Une chaîne débutant par une majuscule.

ˆ.{4,6}\.txt$ Quatre à 6 caractères suivis de “.txt“

ˆ[A − Z].*\.txt$ Une chaîne débutant par une majuscule et finissant par ”.txt“

ˆ$ Une chaîne de caractères vide.

ˆ[ˆ0 − 9]*\.sh$ Une chaîne ne contenant pas de chiffres et se terminant par ”.sh“ Exercice

● En utilisant grep (general regular expression processor) construire une expression régulière permettant de récupérer, dans le fichier hg19_exons_merged.bed, les lignes dont la colonne 1contient les chaînes de caractères chr1, chr2 et chr9 (et pas d’autres chromosomes quoi que puisse contenir le fichier).

● En utilisant awk et un pattern, construire une expression régulière permettant de récupérer, dans le fichier hg19_exons_merged.bed, les lignes dont la colonne 1 contient chr1, chr2 et chr9 (et rien d’autre quoi que puisse contenir le fichier). Solutions

# grep chr1, chr2 et chr9 (et rien d’autre !) # Notez l’utilisation de -P (perl) pour avoir un langage d’expression régulière étendu # utile ici pour la prise en compte de \t. Ne fonctionne pas sou mac. root@vm: grep -P “^chr[123]\t” hg19_exons_merged.bed

# awk chr1, chr2 et chr9 (et rien d’autre !) root@vm: awk ' $1 ~/^chr[123]$/ {print}' hg19_exons_merged.bed Merci