Rui:RNAseq analysis on Hiseq111005: Difference between revisions

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==RNAseq analysis on Hiseq111005==
==RNAseq analysis on Hiseq111005==


===Data===
===Comparison of program===
* 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 w/o G correction===
====Tophat mapping====
=====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
 
=====meangenemachine=====
# 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
# 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 &
 
 
=====bowtie.left_kept_reads.fixmap.log=====
* As proxy of mapping rate.
* Two issues: 1. reads spinning multiple exons can not be included 2. clonal reads included
* Total alignment can be calculated:
samtools flagstat accepted_hits.bam
awk '{print $1}' accepted_hits.bam | wc -l
* Total reads can be estimated as
awk '{print $1}' accepted_hits.bam | sort | uniq | wc -l
* However, total reads reach to 95% of total processed reads???
 
{| {{table}} border=1
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''bowtie.left_kept_reads.fixmap.log'''
| align="center" style="background:#f0f0f0;"|'''Meangenemachine'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Genome-miner'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''report.log'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
|-
| ||||processed||aligned reads ||percentage||failed reads||reads sup. -m||happy splice reads||percentage
| Tophat||||v1.3.1||v1.3.1||||
|-
|-
| E13.5 wt_m1||Indx5||32,990,203||20,649,504||0.6259||12,115,555||225,144||134,070||0.6300
| Bowtie||||0.12.7.0||0.12.7.0||||
|-
|-
| E13.5 wt_f1||Indx6||27,141,595||16,807,921||0.6193||10,178,028||155,646||139,151||0.6244
| Samtools||||0.1.17||0.1.12a||||
|-
|-
| E13.5 wt_m1||Indx9||46,375,424||29,145,806||0.6285||16,865,642||363,976||143,742||0.6316
| BowtieIndex||||(UCSC)||(UCSC)||||(Ensemble)
|-
|-
| E13.5 wt_m2||Indx10||36,895,622||23,534,232||0.6379||13,046,273||315,117||144,857||0.6418
| ||.fa||2011.8.8||2011.8.6||2011.10.27||2011.11.21
|-
|-
| E13.5 wt_f1||Indx11||41,338,700||26,193,747||0.6336||14,856,837||288,116||148,068||0.6372
| ||.ebwt||2011.5.16||2011.5.16||2011.9.27||2011.9.27
|-
|-
| E13.5 wt_f2||Indx12||50,183,035||32,476,668||0.6472||17,347,409||358,958||152,887||0.6502
| Annotation||gene.gtf||2011.5.17||2011.5.17||2011.9.27||2011.9.27
|-
|-
| E13.5 KO_m1||Indx13||34,342,309||22,409,676||0.6525||11,660,995||271,638||133,327||0.6564
| ||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]
|-
|-
| E13.5 KO_f1||Indx14||40,773,127||26,933,424||0.6606||13,505,587||334,116||135,519||0.6639
| cufflinks||||v1.0.3||v1.1.0 (2699)||||
|-
|-
| E13.5 KO_f2||Indx15 ||44,644,927||29,356,424||0.6576||14,891,518||396,985||147,430||0.6609
| cuffdiff||||v1.0.3||v1.1.0 (2699)||||
|}
|}


====Transfer and convert files====
===Data===
=====Transfer files=====
* 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
* Transfer files to genome-miner from meangenemachine
* Libraries to focus on is Indx9|10 (wt_m), Indx11|12 (wt_f), Indx13 (KO_m), Indx14|15 (KO_f)
Transfer files to ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat:
* Data was stored in genome-miner:/media/Ext6T/111005_SN853/RNAseq
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
* make shortcut for input data:  
 
  ln -s /media/Ext6T/111005_SN853/RNAseq Hiseq111005/
ruiliu@meangenemachine-desktop:~/Hiseq111005/tophat_Indx9$
* make a shorcut for output data:
  scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat/tophat_Indx9/* ./
  ln -s /media/Ext4T/DataDrive.backup/RL_Scratch/RNAseq RNAseq/
scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat/tophat_Indx6/* ./
* make copy of sequence file
 
  cp Hiseq111005/* RNAseq/
=====Convert files=====
* combine different files in different lanes into one file for each index:  
* Convert bam to sam in genome-miner
  less *Indx3.txt > Indx3.txt
 
* Transfer files to meangenemachine, as genome-miner is currently busy
  samtools view -h Indx10_accepted_hits.bam > Indx10_accepted_hits.sam
  ssh ruiliu@meangenemachine.dynamic.ucsd.edu
samtools view -h Indx11_accepted_hits.bam > Indx11_accepted_hits.sam
  scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/Indx_seq/* ./
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 &
 
====Cuffdiff====
* Cuffdiff comparison in Ensemble (genome-miner):
* /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf only differs from UCSC gene.gtf in Chr. ID (eg. 8 vs Chr8)
* Dr. Zhang ran a script to change to Chr.ID
 
cd cuffdiff_Ensemble
cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf -N -p 8 -o cuffDiff_wtE13.5f_vs_KOE13.5f ../topha/Indx6_accepted_hits.sam,../tophat/Indx11_accepted_hits.sam,../tophat/Indx12_accepted_hits.sam  ../tophat/Indx14_accepted_hits.sam,../tophat/Indx15_accepted_hits.sam
cuffdiff /GenomeDB/MmGenome/Mus_musculus.NCBIM37.64.gtf -N -p 8 -o cuffDiff_wtE13.5m_vs_KOE13.5m ../tophat/Indx5_accepted_hits.sam,../tophat/Indx9_accepted_hits.sam,../tophat/Indx10_accepted_hits.sam ../tophat/Indx13_accepted_hits.sam
 
* Cuffdiff comparison in UCSC (meangenemachine):
 
cd cuffdiff_UCSC
cuffdiff ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -N -p 8 -o cuffDiff_wtE13.5f_vs_KOE13.5f ../tophat_Indx6/tophat_out/accepted_hits.bam,../tophat_Indx11/tophat_out/accepted_hits.bam,../tophat_Indx12/tophat_out/accepted_hits.bam ../tophat_Indx14/tophat_out/accepted_hits.bam,../tophat_Indx15/tophat_out/accepted_hits.bam
cuffdiff ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -N -p 8 -o cuffDiff_wtE13.5m_vs_KOE13.5m ../tophat_Indx5/tophat_out/accepted_hits.bam,../tophat_Indx9/tophat_out/accepted_hits.bam,../tophat_Indx10/tophat_out/accepted_hits.bam ../tophat_Indx13/tophat_out/accepted_hits.bam
 
* Transfer files in meangenemachine to laptop
scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/cuffdiff_UCSC/cuffDiff_wtE13.5f_vs_KOE13.5f/gene_exp.diff ./Desktop/wtE13.5f_vs_KOE13.5f_gene_exp.diff_UCSC
  scp ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/cuffdiff_UCSC/cuffDiff_wtE13.5m_vs_KOE13.5m/gene_exp.diff ./Desktop/wtE13.5m_vs_KOE13.5m_gene_exp.diff_UCSC
 
* Transfer files in genome-miner to laptop
  scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/cuffdiff_Ensemble/cuffDiff_wtE13.5f_vs_KOE13.5f/gene_exp.diff ./Desktop/wtE13.5f_vs_KOE13.5f_gene_exp.diff_ensemble
scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/cuffdiff_Ensemble/cuffDiff_wtE13.5m_vs_KOE13.5m/gene_exp.diff ./Desktop/wtE13.5m_vs_KOE13.5m_gene_exp.diff_ensemble
 
====Cufflinks====
* genes.FPKM_tracking for each Indx is used to generate a matrix
* prepare a matrix for gene set analysis


cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx5_cufflinks ../tophat_Indx5/tophat_out/accepted_hits.sam
===Tophat_10.25.11 w/o G correction===
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx6_cufflinks ../tophat_Indx6/tophat_out/accepted_hits.sam &
*[[Rui:Tophat mapping|Tophat mapping]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx9_cufflinks ../tophat_Indx9/tophat_out/accepted_hits.sam &
*[[Rui:Transfer and convert files|Transfer and convert files]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx10_cufflinks ../tophat_Indx10/tophat_out/accepted_hits.sam &
*[[Rui:Cuffdiff|Cuffdiff]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx11_cufflinks ../tophat_Indx11/tophat_out/accepted_hits.sam &
*[[Rui:Cufflinks_10.26.11|Cufflinks_10.26.11]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx12_cufflinks ../tophat_Indx12/tophat_out/accepted_hits.sam &
*[[Rui:Matrix|Matrix]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx13_cufflinks ../tophat_Indx13/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx14_cufflinks ../tophat_Indx14/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx15_cufflinks ../tophat_Indx15/tophat_out/accepted_hits.sam &


nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx3_cufflinks ../tophat_Indx3/tophat_out/accepted_hits.sam &
===Tophat_10.27.11 w/ G correction===
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx4_cufflinks ../tophat_Indx4/tophat_out/accepted_hits.sam &
*[[Rui:Tophat mapping 10.27.11|Tophat mapping 10.27.11]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx7_cufflinks ../tophat_Indx7/tophat_out/accepted_hits.sam &
*[[Rui:Cufflinks 10.28.11|Cufflinks 10.28.11]]
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx8_cufflinks ../tophat_Indx8/tophat_out/accepted_hits.sam &
*[[Rui:Matrix 10.30.11|Matrix 10.30.11]]
*[[Rui:Cufflinks_11.7.11|Cufflinks_11.7.11 w/ G annotation]]


* Transfer to genome-miner
==Repeat RNAseq analysis on Hiseq111005 from 11.10.11==
nohup scp -r ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/cufflinks_UCSC ./cufflinks_UCSC
* Lesson 1: Use exactly same VERSION of programs/softwares to do analysis on ALL data in ONE computer
* 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]]


===Matrix===
* Modify Dr. Zhang's script by replacing folder names (such as “tophat_Mm_Indx3” -> “Indx3_cufflinks)
* Under cufflinks_UCSC folder, nano and paste modified script in, save "Combine_geneTracking.pl" (automatically save as -rw-r--r-- in current directory)
* Executive: chomd 755 Combine_geneTracking.pl
* Run: ./Combine_geneTracking.pl > gene_expression_matrix.txt


Problem: I ran tophat w/o G correction, so that output of cufflinks (genes.FPKM_tracking) has no gene annotation. Have to repeat tophat with G correction
==Tophat against Ensemble on 11.14.11==
*[[Rui:Tophat_11.14.11|Rui:Tophat_11.14.11]]
*[[Rui:Tophat_11.21.11|Rui:Tophat_11.21.11]]

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]