Rui:RNAseq analysis on Hiseq111005: Difference between revisions
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===Data=== | ===Data=== | ||
* RNA libraries were used for HL101 run, | * 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
- 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
- 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
- tophat with G correction for major mRNA
- 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