Rui:RNAseq analysis on Hiseq111005: Difference between revisions

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  nohup ~/bin/cufflinks-1.1.0.Linux_x86_64/cufflinks -g ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf accepted_hits.sam &
  nohup ~/bin/cufflinks-1.1.0.Linux_x86_64/cufflinks -g ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf accepted_hits.sam &
=====comparison of genes.fpkm_tracking=====
* count cufflinks genes ID:
cd ../Indx3_cufflinks/
cd ../Indx4_cufflinks/
cd ../Indx5_cufflinks/
cd ../Indx6_cufflinks/
cd ../Indx7_cufflinks/
cd ../Indx8_cufflinks/
cd ../Indx9_cufflinks/
cd ../Indx10_cufflinks/
cd ../Indx11_cufflinks/
cd ../Indx12_cufflinks/
cd ../Indx13_cufflinks/
cd ../Indx14_cufflinks/
cd ../Indx15_cufflinks/
awk '{print $5}' genes.fpkm_tracking | sort | uniq | wc -l > count_genes.fpkm_tracking
less count_genes.fpkm_tracking
* table
There are no much difference in table organization or genes calling
The only difference in gene numbers, I believe, is due to the latest gtf I downloaded in genome-minor which leads to more annotations.
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''tophat w/o G'''
| align="center" style="background:#f0f0f0;"|'''tophat w G'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| cufflinks_Indx3||21266||21913||genome-miner
|-
| cufflinks_Indx4||21250||21896||genome-miner
|-
| cufflinks_Indx5||21238||21881||genome-miner
|-
| cufflinks_Indx6||21253||21896||genome-miner
|-
| cufflinks_Indx7||21348||21344||meangenemachine
|-
| cufflinks_Indx8||21210||21212||meangenemachine
|-
| cufflinks_Indx9||21198||21196||meangenemachine
|-
| cufflinks_Indx10||21209||21221||meangenemachine
|-
| cufflinks_Indx11||21200||21208||meangenemachine
|-
| cufflinks_Indx12||21155||21805||genome-miner
|-
| cufflinks_Indx13||21290||21292||meangenemachine
|-
| cufflinks_Indx14||21257||21255||meangenemachine
|-
| cufflinks_Indx15||21158||21159||meangenemachine
|}

Revision as of 22:01, 31 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 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
  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 &


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???
' ' bowtie.left_kept_reads.fixmap.log ' ' ' ' report.log '
processed aligned reads percentage failed reads reads sup. -m happy splice reads percentage
E13.5 wt_m1 Indx5 32,990,203 20,649,504 0.6259 12,115,555 225,144 134,070 0.6300
E13.5 wt_f1 Indx6 27,141,595 16,807,921 0.6193 10,178,028 155,646 139,151 0.6244
E13.5 wt_m1 Indx9 46,375,424 29,145,806 0.6285 16,865,642 363,976 143,742 0.6316
E13.5 wt_m2 Indx10 36,895,622 23,534,232 0.6379 13,046,273 315,117 144,857 0.6418
E13.5 wt_f1 Indx11 41,338,700 26,193,747 0.6336 14,856,837 288,116 148,068 0.6372
E13.5 wt_f2 Indx12 50,183,035 32,476,668 0.6472 17,347,409 358,958 152,887 0.6502
E13.5 KO_m1 Indx13 34,342,309 22,409,676 0.6525 11,660,995 271,638 133,327 0.6564
E13.5 KO_f1 Indx14 40,773,127 26,933,424 0.6606 13,505,587 334,116 135,519 0.6639
E13.5 KO_f2 Indx15 44,644,927 29,356,424 0.6576 14,891,518 396,985 147,430 0.6609

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

  • 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
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx6_cufflinks ../tophat_Indx6/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx9_cufflinks ../tophat_Indx9/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx10_cufflinks ../tophat_Indx10/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx11_cufflinks ../tophat_Indx11/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx12_cufflinks ../tophat_Indx12/tophat_out/accepted_hits.sam &
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 &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx4_cufflinks ../tophat_Indx4/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx7_cufflinks ../tophat_Indx7/tophat_out/accepted_hits.sam &
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf -o Indx8_cufflinks ../tophat_Indx8/tophat_out/accepted_hits.sam &
  • Transfer to genome-miner
nohup scp -r ruiliu@meangenemachine.dynamic.ucsd.edu:~/Hiseq111005/cufflinks_UCSC ./cufflinks_UCSC

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: chmod 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 w/ G correction

Tophat mapping

  • Tophat against UCSC.gtf
nohup tophat -p 6 --solexa1.3-quals -o Indx15_tophat-G -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx15.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx14_tophat-G -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx14.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx13_tophat-G -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx13.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx12_tophat-G -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx12.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx11_tophat-G -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx11.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx10_tophat-G -G ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx10.txt &
  • genome-miner
Need to download UCSC dataset from iGenome to genome-miner
command: [1]
nohup tophat -p 6 --solexa1.3-quals -o Indx3_tophat-G -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx3.txt &
Others using newly download iGenome on genome-miner (~1hr download with wget, tar 20min) start at 1:40pm
nohup tophat -p 6 --solexa1.3-quals -o Indx4_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx4.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx5_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx5.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx6_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx6.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx7_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx7.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx8_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx8.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx9_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx9.txt &
nohup tophat -p 6 --solexa1.3-quals -o Indx12_tophat-G -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx12.txt &

cufflinks

  • Meangenemachine
nohup samtools view -h ../Indx7_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx8_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx9_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx10_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx11_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx13_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx14_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup samtools view -h ../Indx15_tophat-G/accepted_hits.bam > accepted_hits.sam &
cd ~/Hiseq111005/Indx7/Indx7_cufflinks
cd ~/Hiseq111005/Indx8/Indx8_cufflinks
cd ~/Hiseq111005/Indx9/Indx9_cufflinks
cd ~/Hiseq111005/Indx10/Indx10_cufflinks
cd ~/Hiseq111005/Indx11/Indx11_cufflinks
cd ~/Hiseq111005/Indx13/Indx13_cufflinks
cd ~/Hiseq111005/Indx14/Indx14_cufflinks
cd ~/Hiseq111005/Indx15/Indx15_cufflinks
nohup cufflinks -g ~/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf accepted_hits.sam &
  • Genome-miner
mkdir ~/RNAseq//Hiseq111005/Indx3/Indx3_cufflinks
cd ~/RNAseq//Hiseq111005/Indx3/Indx3_cufflinks
nohup samtools view -h ../Indx3_tophat-G/accepted_hits.bam > accepted_hits.sam &
mkdir ~/RNAseq//Hiseq111005/Indx4/Indx4_cufflinks
cd ~/RNAseq//Hiseq111005/Indx4/Indx4_cufflinks
nohup samtools view -h ../Indx4_tophat-G/accepted_hits.bam > accepted_hits.sam &
mkdir ~/RNAseq//Hiseq111005/Indx5/Indx5_cufflinks
cd ~/RNAseq//Hiseq111005/Indx5/Indx5_cufflinks
nohup samtools view -h ../Indx5_tophat-G/accepted_hits.bam > accepted_hits.sam &
mkdir ~/RNAseq//Hiseq111005/Indx6/Indx6_cufflinks
cd ~/RNAseq//Hiseq111005/Indx6/Indx6_cufflinks
nohup samtools view -h ../Indx6_tophat-G/accepted_hits.bam > accepted_hits.sam &
mkdir ~/RNAseq//Hiseq111005/Indx12/Indx12_cufflinks
cd ~/RNAseq//Hiseq111005/Indx6/Indx6_cufflinks
nohup samtools view -h ../Indx6_tophat-G/accepted_hits.bam > accepted_hits.sam &
nohup ~/bin/cufflinks-1.1.0.Linux_x86_64/cufflinks -g ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf accepted_hits.sam &
comparison of genes.fpkm_tracking
  • count cufflinks genes ID:
cd ../Indx3_cufflinks/
cd ../Indx4_cufflinks/
cd ../Indx5_cufflinks/
cd ../Indx6_cufflinks/
cd ../Indx7_cufflinks/
cd ../Indx8_cufflinks/
cd ../Indx9_cufflinks/
cd ../Indx10_cufflinks/
cd ../Indx11_cufflinks/
cd ../Indx12_cufflinks/
cd ../Indx13_cufflinks/
cd ../Indx14_cufflinks/
cd ../Indx15_cufflinks/
awk '{print $5}' genes.fpkm_tracking | sort | uniq | wc -l > count_genes.fpkm_tracking
less count_genes.fpkm_tracking
  • table
There are no much difference in table organization or genes calling
The only difference in gene numbers, I believe, is due to the latest gtf I downloaded in genome-minor which leads to more annotations.
' tophat w/o G tophat w G '
cufflinks_Indx3 21266 21913 genome-miner
cufflinks_Indx4 21250 21896 genome-miner
cufflinks_Indx5 21238 21881 genome-miner
cufflinks_Indx6 21253 21896 genome-miner
cufflinks_Indx7 21348 21344 meangenemachine
cufflinks_Indx8 21210 21212 meangenemachine
cufflinks_Indx9 21198 21196 meangenemachine
cufflinks_Indx10 21209 21221 meangenemachine
cufflinks_Indx11 21200 21208 meangenemachine
cufflinks_Indx12 21155 21805 genome-miner
cufflinks_Indx13 21290 21292 meangenemachine
cufflinks_Indx14 21257 21255 meangenemachine
cufflinks_Indx15 21158 21159 meangenemachine