HPCwiki - User contributions [en]

RNA-seq analysis

2014-04-13T21:16:23Z

Madse001: Created page with " === Typical commands used for analyzing RNA-seq with Tophat (including Bowtie2 as aligner).=== * Examples are RNA-seq (stranded) from pig aligned against the pig reference ..."

=== Typical commands used for analyzing RNA-seq with Tophat (including Bowtie2 as aligner).===

* Examples are RNA-seq (stranded) from pig aligned against the pig reference genome (''S. scrofa'' - 10.2)

* Tophat, Bowtie2, Picard and GATK need to be in PATH or loaded as modules (e.g.: module load SHARED/bowtie/2-2.2.1; module load SHARED/tophat/2.0.11)
* Bowtie2 index (made with bowtie2_build) of reference genome (need only to be made ones)
* PCR duplicates removed with Picard
* For allelic expression and rna-editing re-aligning with GATK

===For expression analysis===
* Allowing multiply hits (20 times)
<source lang='bash'>
#!/bin/bash
#SBATCH --time=2-12:00:00
#SBATCH -n 1
#SBATCH -c 16
#SBATCH --output=output_%j.txt
#SBATCH --error=error_output_%j.txt
#SBATCH --job-name=AG_tophat
#SBATCH --partition=ABGC_Low
#SBATCH --mem=20000

#Setting java tmp for slurm
export _JAVA_OPTIONS=-Djava.io.tmpdir=/lustre/scratch/WUR/ABGC/madse001/tmp

#Brain_fontalloop
#Tophat alignment
tophat -p 16 -G ../ssc10_2_ens/Sscrofa10.2.68_ens_Tophat.gtf -M --rg-id agLW_BRfl --rg-sample ag_LW_BRfl --rg-platform Illumina --keep-fasta-order --read-realign-edit-dist 0 -r 120 --library-type fr-firststrand \
-o tophat2_agLW_BRfl_g20_peO_reAln_ens_RG_M --mate-std-dev 250 ../ssc10_2_ens/ssc10_2_ens agLW_BRfl_read1.trimmed.final.fastq.gz agLW_BRfl_read2.trimmed.final.fastq.gz

#Rename Tophat alignment file
mv tophat2_agLW_BRfl_g20_peO_reAln_ens_RG_M/accepted_hits.bam tophat2_agLW_BRfl_g20_peO_reAln_ens_RG_M/agLW_BRfl_g20_peO_reAln_ens_RG_M.bam

#Remove PCR duplicates with Picard MarkDuplicates
java -Xms16g -jar ~/bin/MarkDuplicates.jar I=tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M.bam O=tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bam \
M=tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M.bam.out REMOVE_DUPLICATES=true

</source>
===For allelic expression and rna-editing analysis===
* Unique alignment only
<source lang='bash'>
#!/bin/bash
#SBATCH --time=2-12:00:00
#SBATCH -n 1
#SBATCH -c 16
#SBATCH --output=output_%j.txt
#SBATCH --error=error_output_%j.txt
#SBATCH --job-name=AG_tophat
#SBATCH --partition=ABGC_Low
#SBATCH --mem=20000

#Setting java tmp for slurm
export _JAVA_OPTIONS=-Djava.io.tmpdir=/lustre/scratch/WUR/ABGC/madse001/tmp

#Brain_fontalloop
#Tophat alignment
tophat -p 16 -g 1 -G ../ssc10_2_ens/Sscrofa10.2.68_ens_Tophat.gtf -M --rg-id agLW_BRfl --rg-sample ag_LW_BRfl --rg-platform Illumina --keep-fasta-order --read-realign-edit-dist 0 -r 120 --library-type fr-firststrand \
-o tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M --mate-std-dev 250 ../ssc10_2_ens/ssc10_2_ens agLW_BRfl_read1.trimmed.final.fastq.gz agLW_BRfl_read2.trimmed.final.fastq.gz

#Rename Tophat alignment file
mv tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/accepted_hits.bam tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M.bam

#Remove PCR duplicates with Picard
java -Xms16g -jar ~/bin/MarkDuplicates.jar I=tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M.bam O=tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bam \
M=tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M.bam.out REMOVE_DUPLICATES=true

#Index BAM file for GATK analysis
samtools index tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bam tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bai

#Re-align with GATK
java -Xms16g -jar ~/bin/GenomeAnalysisTK.jar -T RealignerTargetCreator -R ../ssc10_2_ens/ssc10_2_ens.fa -I tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bam \
-o tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/realigner.intervals
java -Xms16g -jar ~/bin/GenomeAnalysisTK.jar -T IndelRealigner -R ../ssc10_2_ens/ssc10_2_ens.fa -I tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bam \
-targetIntervals tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/realigner.intervals -o tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp_reG.bam

#Index final BAM file
samtools index tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp_reG.bam tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp_reG.bai

#Remove tmp bam (bai) file
rm tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bam
rm tophat2_agLW_BRfl_g1_peO_reAln_ens_RG_M/agLW_BRfl_g1_peO_reAln_ens_RG_M_RDp.bai

</source>
===Some notes on Tophat options:===
* -g (number of alignments, default 20)
* -M (prefilter-multihits against genome)
* --rg-id; --rg-sample; --keep-fasta-order (needed for many downstream analysis like Picard and GATK)

Bioinformatics tips tricks workflows

2014-04-13T20:06:15Z

Madse001: /* A list of tutorials, workflows, and recipes */

This page is intended as a portal to pages concerning best practices, workflows and pipelines, and other protocols (including scripts).

== A list of tutorials, workflows, and recipes ==
* [[Mapping_reads_with_Mosaik | Mapping Illumina GA2/HiSeq reads to the Sus scrofa genome assembly]]
* [[convert_fastq_to_fasta | A Perl script to convert fastq to fasta file format]]
* [[Mapping Pair-end reads with Stampy]]
* [[making_slices_from_BAM_files | Create slices from a collection of BAM files ]]
* [[Setting_up_Python_virtualenv | Setting up and using a virtual environment for Python3 ]]
* [[ssh_without_password | ssh without password]]
* [[Create_shortcut_log-in_command | Create a shortcut for the ssh log-in command]]
* [[Installing_R_packages_locally | Installing R packages locally]]
* [[command_line_tricks_for_manipulating_fastq | Command-line tricks for manipulating fastq files]]
* [[assemble_mitochondrial_genomes_from_short_read_data | Assemble mitochondrial genomes from whole-genome short-read data]]
* [[1000Bulls_mapping_pipeline_at_ABGC | 1000 Bulls mapping pipeline at ABGC]]
* [[ABGSA | Animal Breeding and Genomics Sequence Archives (ABGSA)]]
* [[Short_read_mapping_pipeline_pig | Pig mapping pipeline at ABGC]]
* [[Extract_noncall_snps_from_soy | Extract a set of pig SNPs not called in a control sample (soybean)]]
* [[calculate_corrected_theta_from_resequencing_data | Calculate nucleotide diversity (theta) corrected for sequencing depth]]
* [[RNA-seq analysis | RNA-seq analysis with Tophat]]

== See also ==
* [[B4F_cluster | B4F Cluster]]

== External links ==
* [http://en.wikipedia.org/wiki/Help:Cheatsheet Help with editing Wiki pages]

Globally installed software

2014-03-17T14:04:55Z

Madse001: /* Available as global SHARED modules */

== Available as modules ==

* gcc/4.8.1
* python/2.7.6
* python/3.3.3
* R/3.0.2

== Globally installed on all nodes ==

* Perl5.10
* pigz
* Python2.6
* BioPerl v1.61
* [http://samtools.sourceforge.net/tabix.shtml bgzip]
* [http://samtools.sourceforge.net/tabix.shtml tabix v0.2.5]

== Available as global SHARED modules ==
Software can be deposited in:
/cm/shared/apps/SHARED/

Modules can be found in:
/cm/shared/modulefiles/SHARED/

{| width="90%"
|- valign="top"
| width="30%" |
* [[allpathslg_48961 | ALLPATHS-LG/48961]]
* [[augustus_2.7 | augustus/2.7]]
* [[bedtools2.18 | bedtools/2.18.0]]
* [[BLAST | BLAST+/2.2.28]]
* [[blat_v35 |blat/v35]]
* [[bowtie2_v2.2.1 | bowtie/2-2.2.1]]
* [[bowtie1_v1.0.0 | bowtie/1-1.0.0]]
* [[bwa_5.9 | bwa/0.5.9]]
* [[bwa_7.5 | bwa/0.7.5a]]
* [[cegma_2.4 | cegma/2.4]]

| width="30%" |

* [[Cufflinks | cufflinks/2.1.1]]
* [[exonerate_2.2.0 | exonerate/2.2.0-x86_64]]
* [[geneid_1.4.4 | geneid/1.4.4]]
* [[genewise_2.2.3 | genewise/2.2.3-rc7]]
* [[gmap_2014-01-21 | gmap/2014-01-21]]
* [[hmmer_3.1 | hmmer/3.1b1]]
* [[jellyfish_2.1.1 | jellyfish/2.1.1]]
* [[MAFFT_7.130 | MAFFT/7.130]]
* [[maker_2.2.8 | maker/2.28]]
* [[Muscle_3.8.31 | muscle/3.8.31]]
* [[Plink_1.07 | Plink/1.07]]

| width="30%" |

* [[Provean_1.1.3 | provean/1.1.3]]
* [[RepeatMasker_4.0.3 | RepeatMasker/4.0.3]]
* [[RepeatModeler_1.0.7 | RepeatModeler/1.0.7]]
* [[RAxML8.0.0 | RAxML/8.0.0]]
* [[samtools v0.1.12a | samtools/0.1.12a]]
* [[samtools v0.1.19 | samtools/0.1.19]]
* [[snap | snap/2013-11-29]]
* [[soapdenovo2_r240 | SOAPdenovo2/r240]]
* [[TopHat_2.0.11 | tophat/2.0.11]]
* [[Trinity_r20131110 | Trinity/r20131110]]

|}

== Adding a custom module directory to your environment ==
To allow the <code>module</code> program to find the custom module directory, the location of that directory has to be added to <code>MODULEPATH</code> variable.

<source lang='bash'>
export MODULEPATH=$MODULEPATH:/cm/shared/modulefiles/SHARED
</source>
This can be made permanent by adding this line of code to the <code>.bash_profile</code> file in the root of your home folder. To then load the modified <code>MODULEPATH</code> variable you have to load <code>.bash_profile</code> again:
<source lang='bash'>
source .bash_profile
</source>
This needs to be done only for terminals that are already open. Next time you login, <code>.bash_profile</code> will be loaded automatically.

You can check if the modules are found.
<source lang='bash'>
module avail
</source>
This should give output that includes something similar to this:

---------------------------------------- /cm/shared/modulefiles/SHARED/ -----------------------------------------
ALLPATHS-LG/48961 bwa/0.7.5a jellyfish/2.1.1 RepeatMasker/4.0.3
augustus/2.7 cegma/2.4 MAFFT/7.130 RepeatModeler/1.0.7
bedtools/2.18.0 cufflinks/2.1.1 maker/2.28 samtools/0.1.12a
BLAST+/2.2.28 exonerate/2.2.0-x86_64 muscle/3.8.31 samtools/0.1.19
blat/v35 geneid/1.4.4 plink/1.07 snap/2013-11-29
bowtie/2-2.2.1 genewise/2.2.3-rc7 provean/1.1.3 SOAPdenovo2/r240
bwa/0.5.9 hmmer/3.1b1 RAxML/8.0.0 tophat/2.0.11

== See also ==
* [[Main_Page#Using_the_B4F_Cluster | Using the B4F Cluster]]
* [[Domain_specific_software_on_B4Fcluster_installation_by_users | Installing domain specific software: installation by users]]
* [[Setting local variables]]
* [[Installing_R_packages_locally | Installing R packages locally]]
* [[Setting_up_Python_virtualenv | Setting up and using a virtual environment for Python3 ]]
* [[ABGC_modules | modules specific for ABGC ]]

== External links ==

User:Madse001

2014-03-16T21:42:56Z

Madse001:

==Ole Madsen==

[[File:OM.png|thumb|right|180x120px]]

[http://scholar.google.com/citations?user=licR_NsAAAAJ&hl=nl Google Scholar]

More later

User:Madse001

2014-03-16T21:41:42Z

Madse001:

==Ole Madsen==

[[File:OM.png|thumb|right|180x120px]]

[http://scholar.google.com/citations?user=licR_NsAAAAJ&hl=nl Google Scholar]

File:OM.png

2014-03-16T21:33:00Z

Madse001:

User:Madse001

2014-03-16T21:31:56Z

Madse001:

==Ole Madsen==

[[File:/home/ole/Dropbox/documents/OM.png]]

[http://scholar.google.com/citations?user=licR_NsAAAAJ&hl=nl Google Scholar]

User:Madse001

2014-03-16T21:29:26Z

Madse001: Created page with "==Ole Madsen== [http://scholar.google.com/citations?user=licR_NsAAAAJ&hl=nl Google Scholar]"

==Ole Madsen==

[http://scholar.google.com/citations?user=licR_NsAAAAJ&hl=nl Google Scholar]