Rui:RNAseq analysis on Hiseq111005: Difference between revisions

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  nohup cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.chr.64.gtf -N -p 8 -o 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 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 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 &
  nohup cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.chr.64.gtf -N -p 8 -o 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==
* cuffdiff against Ensemble results in much less DE ~300 in E13.5f, ~100 in E13.5m
* Repeat tophat against Ensemble gtf and Ensemble bowtie Index
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 &

Revision as of 18:48, 14 November 2011

RNAseq analysis on Hiseq111005

Data

  • 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

Tophat_10.27.11 w/ G correction

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

  • Lesson 1: Use exactly same VERSION of programs/softwares to do analysis on ALL data in ONE computer
  • Lesson 2: Random check for comparison

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 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 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 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 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

  • cuffdiff against Ensemble results in much less DE ~300 in E13.5f, ~100 in E13.5m
  • Repeat tophat against Ensemble gtf and Ensemble bowtie Index
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 &