DE expression - Revision history

Derks047 at 08:44, 27 January 2016

2016-01-27T08:44:40Z

← Older revision		Revision as of 10:44, 27 January 2016
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	=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff ===		=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff ===

	Martijn Derks		Author: Martijn Derks

	* Samtools, Trimmomatic, Tophat, Bowtie2, Stringtie, Cufflinks need to be in PATH.		* Samtools, Trimmomatic, Tophat, Bowtie2, Stringtie, Cufflinks need to be in PATH.

Derks047 at 08:44, 27 January 2016

2016-01-27T08:44:30Z

← Older revision		Revision as of 10:44, 27 January 2016
Line 1:		Line 1:
	=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff ===		=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff ===

			Martijn Derks

	* Samtools, Trimmomatic, Tophat, Bowtie2, Stringtie, Cufflinks need to be in PATH.		* Samtools, Trimmomatic, Tophat, Bowtie2, Stringtie, Cufflinks need to be in PATH.

Derks047: /* Remove PCR duplicates and use stringtie to quantify/assemble transcripts */

2016-01-26T12:59:11Z

Remove PCR duplicates and use stringtie to quantify/assemble transcripts

← Older revision		Revision as of 14:59, 26 January 2016
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	for f in $FILES		for f in $FILES
	do		do
	~~/home/NIOO/martijnd/progs_nobackup/samtools-1.3/~~samtools sort $f -o $f.sorted		samtools sort $f -o $f.sorted
	~~/home/NIOO/martijnd/progs_nobackup/samtools-1.3/~~samtools rmdup $f.sorted $f.sorted.rmdup.bam		samtools rmdup $f.sorted $f.sorted.rmdup.bam
	ftrim=${f:0:-18}		ftrim=${f:0:-18}
	name=`echo $f \| cut -d"/" -f2`		name=`echo $f \| cut -d"/" -f2`

Derks047: /* Merge transcriptomes */

2016-01-26T12:54:40Z

Merge transcriptomes

← Older revision		Revision as of 14:54, 26 January 2016
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	</source>		</source>

			===Differential expression with Cuffdiff===
	<source lang='bash'>		<source lang='bash'>
	~~===Differential expression with Cuffdiff===~~
	mkdir -p CUFFDIFF		mkdir -p CUFFDIFF
	cuffdiff -o CUFFDIFF -p 32 -L Egg,Caterpillar,PupaStg1,PupaStg2,PupaStg3,Moth MERGED/merged.gtf \		cuffdiff -o CUFFDIFF -p 32 -L Egg,Caterpillar,PupaStg1,PupaStg2,PupaStg3,Moth MERGED/merged.gtf \

Derks047: /* Differential expression with Cuffdiff */

2016-01-26T12:54:13Z

Differential expression with Cuffdiff

← Older revision		Revision as of 14:54, 26 January 2016
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	</source>		</source>

			<source lang='bash'>
	===Differential expression with Cuffdiff===		===Differential expression with Cuffdiff===
	mkdir -p CUFFDIFF		mkdir -p CUFFDIFF

Derks047: Created page with "=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff === * Samtools, Trimmomatic, Tophat, Bowtie2, S..."

2016-01-26T12:53:16Z

Created page with "=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff === * Samtools, Trimmomatic, Tophat, Bowtie2, S..."

New page

=== Typical commands used for doing a DE expression study using Tophat (including Bowtie2 as aligner), Stringtie and Cuffdiff ===

* Samtools, Trimmomatic, Tophat, Bowtie2, Stringtie, Cufflinks need to be in PATH.
* Use case in wintermoth. 12 samples (1 Egg, 1 Caterpillar, 3x3 Pupa, 1 Moth).

===Trim sequences for quality and adapters===
<source lang='bash'>
DIRS='/data/ngs/AnE/visser_group/Operophtera_brumata/data/rnaseq/raw/*'
mkdir -p TRIM
for d in $DIRS
do
R1=$d/*R1*.fastq.gz
R2=$d/*R2*.fastq.gz
lname=`echo $R1 | cut -d"/" -f10`
echo $lname
mkdir -p TRIM/$lname
cd TRIM/$lname
java -jar ~/progs_nobackup/Trimmomatic-0.35/trimmomatic-0.35.jar PE -phred33 -threads 32 $R1 $R2 $lname\_trimmed_R1.fastq.gz $lname\_trimmed_R1_unpaired.fastq.gz $lname\_trimmed_R2.fastq.gz $lname\_trimmed_R2_unpaired.fastq.gz ILLUMINACLIP:/home/NIOO/martijnd/progs_nobackup/Trimmomatic-0.35/adapters/TruSeq3-PE-2.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 HEADCROP:7 MINLEN:50 >> ../wm_rnaseq_trimmomatic.log
cd ..
done
</source>

===Perform mapping with tophat2===
<source lang='bash'>
mkdir -p MAPPING
mkdir -p assembly/bowtie2-index
bowtie2-build assembly/Obru1.fsa assembly/bowtie2-index/Obru1

DIRS='TRIM/*'
for d in $DIRS
do
log='TRIM/wm_rnaseq_trimmomatic.log'
if [[ "$d" == "$log" ]]; then continue; fi
R1=`echo $d/*R1.fastq.gz`
R2=`echo $d/*R2.fastq.gz`
R1_U=`echo $d/*R1_unpaired.fastq.gz`
R2_U=`echo $d/*R2_unpaired.fastq.gz`
lname=`echo $R1 | cut -d"/" -f2`
cat $R1_U $R2_U > $d/unpaired.fastq.gz
echo $lname
tophat2 -p 32 --read-mismatches 8 --read-gap-length 4 --read-edit-dist 8 -o MAPPING/$lname assembly/bowtie2-index/Obru1 $R1 $R2,$d/unpaired.fastq.gz
done
</source>

===Remove PCR duplicates and use stringtie to quantify/assemble transcripts===
<source lang='bash'>
mkdir -p STRINGTIE
FILES='MAPPING/*/*hits.bam'
for f in $FILES
do
/home/NIOO/martijnd/progs_nobackup/samtools-1.3/samtools sort $f -o $f.sorted
/home/NIOO/martijnd/progs_nobackup/samtools-1.3/samtools rmdup $f.sorted $f.sorted.rmdup.bam
ftrim=${f:0:-18}
name=`echo $f | cut -d"/" -f2`
echo $name
stringtie -p 32 -G assembly/Obru_genes.gff -o STRINGTIE/$name/transcripts.gtf $f.sorted.rmdup.bam
echo STRINGTIE/$name/transcripts.gtf >> cuffmerge_gtf_list.txt
done
</source>

===Merge transcriptomes===
<source lang='bash'>
cuffmerge -o MERGED -g assembly/Obru_genes.gff -s assembly/Obru1.fsa -p 32 STRINGTIE/cuffmerge_gtf_list.txt
</source>

===Differential expression with Cuffdiff===
mkdir -p CUFFDIFF
cuffdiff -o CUFFDIFF -p 32 -L Egg,Caterpillar,PupaStg1,PupaStg2,PupaStg3,Moth MERGED/merged.gtf \
MAPPING/run0183IL_34_Egg/accepted_hits.bam.sorted.rmdup.bam \
MAPPING/run0183IL_rups_502-5/accepted_hits.bam.sorted.rmdup.bam \
MAPPING/run0183IL_pop1/accepted_hits.bam.sorted.rmdup.bam,MAPPING/run0183IL_pop2/accepted_hits.bam.sorted.rmdup.bam,MAPPING/run0183IL_pop3/accepted_hits.bam.sorted.rmdup.bam \
MAPPING/run0183IL_pop4/accepted_hits.bam.sorted.rmdup.bam,MAPPING/run0183IL_pop5/accepted_hits.bam.sorted.rmdup.bam,MAPPING/run0183IL_pop6/accepted_hits.bam.sorted.rmdup.bam \
MAPPING/run0183IL_pop7/accepted_hits.bam.sorted.rmdup.bam,MAPPING/run0183IL_pop8/accepted_hits.bam.sorted.rmdup.bam,MAPPING/run0183IL_pop9/accepted_hits.bam.sorted.rmdup.bam \
MAPPING/run0183IL_5__wintervlinder/accepted_hits.bam.sorted.rmdup.bam
</source>