Rui:Tophat mapping

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Tophat mapping 10.25.11[edit]

genome-miner[edit]
  • 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[edit]
  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[edit]
  • 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