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]
- 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[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 |