Rui:RNAseq analysis on Hiseq111005: Difference between revisions

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===Data===
===Data===
* RNA libraries were used for HL101 run, Indx5-13, [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui_Liu#RNA_libraries_for_Dr._Yi_Zhang_lab
* RNA libraries were used for HL101 run, Indx3-15, [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui_Liu#RNA_libraries_for_Dr._Yi_Zhang_lab
* Libraries to focus on is Indx9|10 (wt_m), Indx11|12 (wt_f), Indx13 (KO_m), Indx14|15 (KO_f)
* Data was stored in genome-miner:/media/Ext6T/111005_SN853/RNAseq
* Data was stored in genome-miner:/media/Ext6T/111005_SN853/RNAseq



Revision as of 23:01, 24 October 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

  • PATH issue from Athurva's note: still not working
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
  • Tophat running on meangenemachine
  1. tophat with G correction for major mRNA
  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