Rui:RNAseq analysis on Hiseq111005

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Revision as of 18:16, 26 October 2011 by >RuiLiu (→‎Transfer files)
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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

genome-miner

  • PATH issue from Athurva's note:
  • Need PATH for bowtie, samtools, as well as GenomeDB??
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ ~kunzhang/softwares/bowtie-latest/
bowtie                bowtie-build-debug    bowtie-inspect        doc/                  indexes/              scripts/              
bowtie-build          bowtie-debug          bowtie-inspect-debug  genomes/              reads/                
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ bowtie
^C
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ ls
nohup.out  tophat_Mm_Indx3
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ echo $PATH
/usr/local/sbin:/usr/local/bin:/usr/sbin:/usr/bin:/sbin:/bin:/usr/games
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ PATH=/home/kunzhang/softwares/
abyss-1.2.5/                            Dindel/                                 QuEST_2.4/
audy-stitch-db9e338/                    dindel-1.01-linux.tar.gz                QuEST_2.4.tar.gz
audy-stitch-db9e338.tar.gz              dindel-1.01-python/                     README.txt
beagle.jar                              GenomeAnalysisTK-1.0.3864/              samtools-0.1.12a/
blast-2.2.20/                           GenomeAnalysisTK-1.0.4905/              samtools-0.1.7_x86_64-linux/
blast-2.2.20-x64-linux.tar.gz           GenomeAnalysisTK-1.0.5083/              samtools-0.1.7_x86_64-linux.tar
blat_34/                                GenomeAnalysisTK-latest/                samtools-latest/
bowtie-0.12.7/                          GenomeAnalysisTK-latest.tar             SegSeq_1.0.1/
bowtie-latest/                          gm_key_64.tar                           SegSeq_1.0.1.tar.gz
brat-1.2.2/                             greatTools/                             SHERA/
brat-1.2.2.tar.gz                       greatTools.tar.gz                       SHERA_files.tar.gz
bwa-0.5.8c/                             Homo_sapiens_UCSC_hg19.tar.gz           SNVMix2-0.11.8-r3/
bwa-0.5.9/                              human_empty.bam                         SNVMix2-0.11.8-r3.tar.gz
bwa-0.5.9.tar                           impute_v2.1.2_x86_64_static.tgz         soap2.20release/
bwa-latest/                             jksrc/                                  SOAPdenovo_Release1.04/
cgatools-1.3.0.9-docs/                  jksrc.zip                               sratoolkit.2.1.6-centos_linux64/
cgatools-1.3.0.9-docs.tar.gz            macs_1.4.1.deb                          sratoolkit.2.1.6-centos_linux64.tar.gz
cgatools-1.3.0.9-linux-x86_64/          metaGene/                               stampy-1.0.8/
cgatools-1.3.0.9-linux-x86_64.tar.gz    MetaGeneMark_linux64/                   stampy-latest.tgz
cnver-0.7.2/                            MetaGeneMark_linux64.tar.gz             taoliu-MACS-7268e40/
cnver-0.7.2.tar.gz                      mga_ia64.tar                            taoliu-MACS-v2.0.7-11-g7268e40.tar.gz
CNVnator/                               ncbi-blast-2.2.24+/                     tophat-1.2.0.Linux_x86_64/
CNVnator_v0.2.2.zip                     OLB-1.8.0/                              tophat-1.3.1.Linux_x86_64/
cufflinks-1.0.3.Linux_x86_64/           OLB-1.9.3/                              tophat-1.3.1.Linux_x86_64.tar.gz
cufflinks-1.0.3.Linux_x86_64.tar.gz     OLB-1.9.3.tar.tar.gz                    tophat-latest/
cufflinks-1.1.0.Linux_x86_64/           Phrap/                                  trinityrnaseq_r2011-07-13/
cufflinks-1.1.0.Linux_x86_64.tar.gz     Phrap-distrib.tar.Z                     trinityrnaseq_r2011-07-13.tgz
cufflinks-latest/                       phred-dist-020425.c-acd.tar.Z           velvet_1.0.18/
Cython-0.15/                            picard-tools-1.38/                      
Cython-0.15.tar.gz                      picard-tools-latest/                    
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ PATH=/home/kunzhang/softwares/^C
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ PATH=/home/kunzhang/softwares/bowtie-latest:$PATH /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx3 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx3.txt
  • Add both bowtie and samtools to PATH:
PATH=/home/kunzhang/softwares/bowtie-latest:$PATH PATH=/home/kunzhang/softwares/samtools-latest:$PATH /home/kunzhang/softwares/tophat-latest/tophat -p 6 --solexa1.3-quals -o tophat_Indx9 /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx9.txt
  • Dr. Zhang fixed the problem of PATH, then simply run tophat under my directory:

tophat -p 6 --solexa1.3-quals -o tophat_Indx9 /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx9.txt

Tophat running on meangenemachine

  1. tophat with G correction for major mRNA, based on data analysis on HL098, mapped reads from G correction are more (~100k) than ones w/o G correction
  2. Tophat without G correction for non-coding RNAs, plan as follows:
Here is my recommendation for your analysis:
(1)    Perform tophat mapping without any gene model.
(2)    Perform cuffdiff analysis using the UCSC gene model (like you did before) to look at protein coding genes.
(3)    Perform cuffdiff analysis using the Ensembl gene model to look at both coding and noncoding genes (you can compare the coding ones between (2) and (3) to check the consistency.
(4)    For functional annotation of LincRNAs that we don’t know too much about, perhaps you can use the GREAT analysis (http://great.stanford.edu/public/cgi-bin/greatWeb.php) , because most of the LincRNAs act in a cis- manner.
Ensemble reference
/GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf
mkdir tophat_Indx15
nohup tophat -p 6 --solexa1.3-quals ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx15.txt &

mkdir tophat_Indx15-g
nohup tophat -p 6 --solexa1.3-quals -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx15.txt &

Transfer and convert files

Transfer files

  • Transfer files to genome-miner from meangenemachine
Transfer files to ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat:
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx15/tophat_out/accepted_hits.bam ./Indx15_accepted_hits.bam
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx14/tophat_out/accepted_hits.bam ./Indx14_accepted_hits.bam
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx13/tophat_out/accepted_hits.bam ./Indx13_accepted_hits.bam
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx12/tophat_out/accepted_hits.bam ./Indx12_accepted_hits.bam
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx11/tophat_out/accepted_hits.bam ./Indx11_accepted_hits.bam
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx10/tophat_out/accepted_hits.bam ./Indx10_accepted_hits.bam
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/tophat_Indx5/tophat_out/accepted_hits.bam ./Indx5_accepted_hits.bam
  • Transfer files from genome-miner to meangenemachine
Transfer files to meangenemachine:
ruiliu@meangenemachine-desktop:~/Hiseq111005/tophat_Indx9$ 
scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat/tophat_Indx9/* ./
scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat/tophat_Indx6/* ./

Convert files

  • Convert bam to sam in genome-miner
samtools view -h Indx10_accepted_hits.bam > Indx10_accepted_hits.sam
samtools view -h Indx11_accepted_hits.bam > Indx11_accepted_hits.sam
nohup samtools view -h Indx12_accepted_hits.bam > Indx12_accepted_hits.sam &
nohup samtools view -h Indx13_accepted_hits.bam > Indx13_accepted_hits.sam
nohup samtools view -h Indx14_accepted_hits.bam > Indx14_accepted_hits.sam
nohup samtools view -h Indx15_accepted_hits.bam > Indx15_accepted_hits.sam
nohup samtools view -h Indx5_accepted_hits.bam > Indx5_accepted_hits.sam
nohup samtools view -h Indx6_accepted_hits.bam > Indx6_accepted_hits.sam
nohup samtools view -h Indx9_accepted_hits.bam > Indx9_accepted_hits.sam
  • Convert bam to sam in meangenemachine
nohup samtools view -h ./tophat_Indx10/tophat_out/accepted_hits.bam > ./tophat_Indx10/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx11/tophat_out/accepted_hits.bam > ./tophat_Indx11/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx12/tophat_out/accepted_hits.bam > ./tophat_Indx12/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx13/tophat_out/accepted_hits.bam > ./tophat_Indx13/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx14/tophat_out/accepted_hits.bam > ./tophat_Indx14/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx15/tophat_out/accepted_hits.bam > ./tophat_Indx15/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx5/tophat_out/accepted_hits.bam > ./tophat_Indx5/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx6/tophat_out/accepted_hits.bam > ./tophat_Indx6/tophat_out/accepted_hits.sam &
nohup samtools view -h ./tophat_Indx9/tophat_out/accepted_hits.bam > ./tophat_Indx9/tophat_out/accepted_hits.sam &