Rui:RNAseq analysis on Hiseq111005: Difference between revisions

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==RNAseq analysis on Hiseq111005==
==RNAseq analysis on Hiseq111005==
===Comparison of program===
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Meangenemachine'''
| align="center" style="background:#f0f0f0;"|'''Genome-miner'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| 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||[http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:Tophat_11.21.11#Tophat_11.21.11]
|-
| cufflinks||||v1.0.3||v1.1.0 (2699)||||
|-
| cuffdiff||||v1.0.3||v1.1.0 (2699)||||
|}


===Data===
===Data===
Line 18: Line 48:
  scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/Indx_seq/* ./
  scp ruiliu@genome-miner:~/RNAseq/Hiseq111005/Indx_seq/* ./


===Tophat===
===Tophat_10.25.11 w/o G correction===
====genome-miner====
*[[Rui:Tophat mapping|Tophat mapping]]
* PATH issue from Athurva's note:
*[[Rui:Transfer and convert files|Transfer and convert files]]
* Need PATH for bowtie, samtools, as well as GenomeDB??
*[[Rui:Cuffdiff|Cuffdiff]]
 
*[[Rui:Cufflinks_10.26.11|Cufflinks_10.26.11]]
ruiliu@genome-miner:~/RNAseq/Hiseq111005/tophat$ ~kunzhang/softwares/bowtie-latest/
*[[Rui:Matrix|Matrix]]
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====
# 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 &
 
===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
 
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 &
 


===Cuffdiff===
===Tophat_10.27.11 w/ G correction===
* Cuffdiff comparison in Ensemble:
*[[Rui:Tophat mapping 10.27.11|Tophat mapping 10.27.11]]
*[[Rui:Cufflinks 10.28.11|Cufflinks 10.28.11]]
*[[Rui:Matrix 10.30.11|Matrix 10.30.11]]
*[[Rui:Cufflinks_11.7.11|Cufflinks_11.7.11 w/ G annotation]]


cd cuffdiff_Ensemble
==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
*[[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]]


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
==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]