Rui:RNAseq analysis on Hiseq111005: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>RuiLiu
>RuiLiu
 
(9 intermediate revisions by the same user not shown)
Line 18: Line 18:
| BowtieIndex||||(UCSC)||(UCSC)||||(Ensemble)
| BowtieIndex||||(UCSC)||(UCSC)||||(Ensemble)
|-
|-
| ||.fa||2011.8.8||2011.8.6||2011.10.27||2011.8.6
| ||.fa||2011.8.8||2011.8.6||2011.10.27||2011.11.21
|-
|-
| ||.ebwt||2011.5.16||2011.5.16||2011.9.27||2011.6.13
| ||.ebwt||2011.5.16||2011.5.16||2011.9.27||2011.9.27
|-
|-
| Annotation||gene.gtf||2011.5.17||2011.5.17||2011.9.27||2011.10.16
| Annotation||gene.gtf||2011.5.17||2011.5.17||2011.9.27||2011.9.27
|-
|-
| ||file||~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/gene.gtf||/GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf||~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/gene.gtf||/GenomeDB/MmGenome
| ||file||~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/gene.gtf||/GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf||~/RNAseq/iGenome/UCSC/Mus_musculus/UCSC/mm9/Annotation/Genes/gene.gtf||[http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:Tophat_11.21.11#Tophat_11.21.11]
|-
|-
| cufflinks||||v1.0.3||v1.1.0 (2699)||||
| cufflinks||||v1.0.3||v1.1.0 (2699)||||
Line 59: Line 59:
*[[Rui:Cufflinks 10.28.11|Cufflinks 10.28.11]]
*[[Rui:Cufflinks 10.28.11|Cufflinks 10.28.11]]
*[[Rui:Matrix 10.30.11|Matrix 10.30.11]]
*[[Rui:Matrix 10.30.11|Matrix 10.30.11]]
 
*[[Rui:Cufflinks_11.7.11|Cufflinks_11.7.11 w/ G annotation]]
===Cufflinks_11.7.11 w/ G annotation===
* Previously, I used -g option which allows reference as the guide
* Dr. Zhang used -G option which excludes any novel transcripts
* To repeat cufflinks in genome-miner by using the following command
nohup /home/kunzhang/softwares/cufflinks-1.1.0.Linux_x86_64/cufflinks -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf -N -p 2 -o Indx13 /home/ruiliu/RNAseq/Hiseq111005/tophat/Indx13_accepted_hits.bam > Indx13.log&
 
Problem need to solve:
# can .bam file directly used for cufflinks?
# is the tracking_id exactly same as gene_id?
 
--- genome-minor, bam file worked!
 
mkdir ~/RNAseq/Hiseq111005/cufflinks-G
nohup /home/kunzhang/softwares/cufflinks-1.1.0.Linux_x86_64/cufflinks -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf -N -p 2 -o Indx13 /home/ruiliu/RNAseq/Hiseq111005/tophat/Indx13_accepted_hits.bam > Indx13.log&
nohup /home/kunzhang/softwares/cufflinks-1.1.0.Linux_x86_64/cufflinks -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf -N -p 2 -o Indx14 /home/ruiliu/RNAseq/Hiseq111005/tophat/Indx14_accepted_hits.bam > Indx14.log&
nohup /home/kunzhang/softwares/cufflinks-1.1.0.Linux_x86_64/cufflinks -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf -N -p 2 -o Indx15 /home/ruiliu/RNAseq/Hiseq111005/tophat/Indx15_accepted_hits.bam > Indx15.log&
 
--- meangenemachine, bam file failed again!
 
nohup cufflinks -G ~/iGenome/ ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -N -p 2 -o Indx13_cufflinks ~/Hiseq111005/Indx13/Indx13_tophat-G/accepted_hits.bam > Indx13.log&


==Repeat RNAseq analysis on Hiseq111005 from 11.10.11==
==Repeat RNAseq analysis on Hiseq111005 from 11.10.11==
* Lesson 1: Use exactly same VERSION of programs/softwares to do analysis on ALL data in ONE computer
* Lesson 1: Use exactly same VERSION of programs/softwares to do analysis on ALL data in ONE computer
* Lesson 2: Random check for comparison
* Lesson 2: Random check for comparison
*[[Rui:Tophat_11.10.11|Tophat_11.10.11]]
*[[Rui:Cufflinks_11.11.11|Cufflinks_11.11.11]]
*[[Rui:Cuffdiff_11.11.11|Cuffdiff_11.11.11]]


===Tophat_11.10.11===
* Genome-minor from 11:15am on 11.10.11
* Tophat 1.3.1
* Re-do on Indx 10, 11, 13, 14, 15, and 3
* move tophat folders into tophat_11.10.11
nohup tophat -p 3 --solexa1.3-quals -o Indx3 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx3.txt &
nohup tophat -p 3 --solexa1.3-quals -o Indx10 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx10.txt &
nohup tophat -p 3 --solexa1.3-quals -o Indx11 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx11.txt &
nohup tophat -p 3 --solexa1.3-quals -o Indx13 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx13.txt &
nohup tophat -p 3 --solexa1.3-quals -o Indx14 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx14.txt &
nohup tophat -p 3 --solexa1.3-quals -o Indx15 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx15.txt &
===Cufflinks_11.11.11===
===Cuffdiff_11.11.11===
* UCSC gtf
nohup cuffdiff ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -N -p 4 -o UCSC_cuffDiff_wtE13.5f_vs_KOE13.5f ../tophat_10.27.11/Indx6/Indx6_tophat/accepted_hits.bam,../tophat_11.10.11/Indx11/Indx11_tophat/accepted_hits.bam,../tophat_10.27.11/Indx12/Indx12_tophat/accepted_hits.bam ../tophat_11.10.11/Indx14/Indx14_tophat/accepted_hits.bam,../tophat_11.10.11/Indx15/Indx15_tophat/accepted_hits.bam &
nohup cuffdiff ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -N -p 4 -o UCSC_cuffDiff_wtE13.5m_vs_KOE13.5m ../tophat_10.27.11/Indx5/Indx5_tophat/accepted_hits.bam,../tophat_10.27.11/Indx9/Indx9_tophat/accepted_hits.bam,../tophat_11.10.11/Indx10/Indx10_tophat/accepted_hits.bam ../tophat_11.10.11/Indx13/Indx13_tophat/accepted_hits.bam &
* Emsemble gtf
nohup cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.chr.64.gtf -N -p 8 -o Ensemble_cuffDiff_wtE13.5f_vs_KOE13.5f ../tophat_10.27.11/Indx6/Indx6_tophat/accepted_hits.bam,../tophat_11.10.11/Indx11/Indx11_tophat/accepted_hits.bam,../tophat_10.27.11/Indx12/Indx12_tophat/accepted_hits.bam ../tophat_11.10.11/Indx14/Indx14_tophat/accepted_hits.bam,../tophat_11.10.11/Indx15/Indx15_tophat/accepted_hits.bam &
nohup cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.chr.64.gtf -N -p 8 -o Ensemble_cuffDiff_wtE13.5m_vs_KOE13.5m ../tophat_10.27.11/Indx5/Indx5_tophat/accepted_hits.bam,../tophat_10.27.11/Indx9/Indx9_tophat/accepted_hits.bam,../tophat_11.10.11/Indx10/Indx10_tophat/accepted_hits.bam ../tophat_11.10.11/Indx13/Indx13_tophat/accepted_hits.bam &


==Tophat against Ensemble on 11.14.11==
==Tophat against Ensemble on 11.14.11==
* cuffdiff against Ensemble results in much less DE ~300 in E13.5f, ~100 in E13.5m
*[[Rui:Tophat_11.14.11|Rui:Tophat_11.14.11]]
* Repeat tophat against Ensemble gtf and Ensemble bowtie Index
*[[Rui:Tophat_11.21.11|Rui:Tophat_11.21.11]]
 
nohup tophat -p 8 --solexa1.3-quals -o Indx15_E_E -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.chr.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx15.txt &
nohup tophat -p 8 --solexa1.3-quals -o Indx15_E_U -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.chr.64.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx15.txt &
 
* Use the original Ensemble gtf, nano "batch command", save as tophat_E_E.sh, sh tophat_E_E.sh
 
nohup tophat -p 2 --solexa1.3-quals -o Indx15 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx15.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx14 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx14.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx13 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx13.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx12 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx12.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx11 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx11.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx10 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx10.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx9 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx9.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx6 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx6.txt &
nohup tophat -p 2 --solexa1.3-quals -o Indx5 -G /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf /GenomeDB/MmGenome/mm9/mm9 ../Indx_seq/Indx5.txt &
 
* cuffdiff_11.20.11
nohup cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf -N -p 10 -o Ensemble_cuffDiff_wtE13.5m_vs_KOE13.5m ../tophat_11.14.11/Indx5/accepted_hits.bam,../tophat_11.14.11/Indx9/accepted_hits.bam,../tophat_11.14.11/Indx10/accepted_hits.bam ../tophat_11.14.11/Indx13/accepted_hits.bam &
nohup cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf -N -p 10 -o Ensemble_cuffDiff_wtE13.5f_vs_KOE13.5f ../tophat_11.14.11/Indx6/accepted_hits.bam,../tophat_11.14.11/Indx11/accepted_hits.bam,../tophat_11.14.11/Indx12/accepted_hits.bam ../tophat_11.14.11/Indx14/accepted_hits.bam,../tophat_11.14.11/Indx15/accepted_hits.bam &

Latest revision as of 18:07, 22 November 2011

RNAseq analysis on Hiseq111005[edit]

Comparison of program[edit]

' ' Meangenemachine Genome-miner ' '
Tophat v1.3.1 v1.3.1
Bowtie 0.12.7.0 0.12.7.0
Samtools 0.1.17 0.1.12a
BowtieIndex (UCSC) (UCSC) (Ensemble)
.fa 2011.8.8 2011.8.6 2011.10.27 2011.11.21
.ebwt 2011.5.16 2011.5.16 2011.9.27 2011.9.27
Annotation gene.gtf 2011.5.17 2011.5.17 2011.9.27 2011.9.27
file ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/gene.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC/Mus_musculus/UCSC/mm9/Annotation/Genes/gene.gtf [1]
cufflinks v1.0.3 v1.1.0 (2699)
cuffdiff v1.0.3 v1.1.0 (2699)

Data[edit]

  • make shortcut for input data:
ln -s /media/Ext6T/111005_SN853/RNAseq Hiseq111005/
  • make a shorcut for output data:
ln -s /media/Ext4T/DataDrive.backup/RL_Scratch/RNAseq RNAseq/
  • make copy of sequence file
cp Hiseq111005/* RNAseq/
  • combine different files in different lanes into one file for each index:
less *Indx3.txt > Indx3.txt
  • Transfer files to meangenemachine, as genome-miner is currently busy
ssh ruiliu@meangenemachine.dynamic.ucsd.edu
scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/Indx_seq/* ./

Tophat_10.25.11 w/o G correction[edit]

Tophat_10.27.11 w/ G correction[edit]

Repeat RNAseq analysis on Hiseq111005 from 11.10.11[edit]


Tophat against Ensemble on 11.14.11[edit]