Rui:RNAseq analysis on HL109

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HL109[edit]

  • Samples: assumed to be HL106 [1]

Data[edit]

  • Transfer files to Indx_seq folder, save as "less" batch command, Indx92,95 and 96 were also used by Noi, repeat less
less /home/ruiliu/SeqStore/111112_HL109/*_Indx88.txt > Indx88.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx89.txt > Indx89.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx90.txt > Indx90.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx91.txt > Indx91.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx92.txt > Indx92.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx93.txt > Indx93.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx94.txt > Indx94.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx95.txt > Indx95.txt
less /home/ruiliu/SeqStore/111112_HL109/*_Indx96.txt > Indx96.txt

Tophat_11.20.11 Mm folder[edit]

  • Using UCSC bowtieIndex and Annotation
  • "tophat" batch command
tophat -p 10 --solexa1.3-quals -o Indx88 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx88.txt 
tophat -p 10 --solexa1.3-quals -o Indx89 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx89.txt 
tophat -p 10 --solexa1.3-quals -o Indx90 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx90.txt 
tophat -p 10 --solexa1.3-quals -o Indx91 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx91.txt 
tophat -p 10 --solexa1.3-quals -o Indx92 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx92.txt
tophat -p 10 --solexa1.3-quals -o Indx93 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx93.txt  
tophat -p 10 --solexa1.3-quals -o Indx94 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx94.txt 
tophat -p 10 --solexa1.3-quals -o Indx95 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx95.txt 
tophat -p 10 --solexa1.3-quals -o Indx96 -G ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome ../Indx_seq/Indx96.txt

Tophat_11.20.11[edit]

Re-download UCSC_Hs package from iGenome[edit]

wget ftp://igenome:G3nom3s4u@ftp.illumina.com/Homo_sapiens/UCSC/hg19/Homo_sapiens_UCSC_hg19.tar.gz
tar -zxvf Homo_sapiens_UCSC_hg19.tar.gz 
BowtieIndex: ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome 
Annotation: ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf

Re-run tophat[edit]

tophat -p 10 --solexa1.3-quals -o Indx88 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx88.txt 
tophat -p 10 --solexa1.3-quals -o Indx89 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx89.txt
tophat -p 10 --solexa1.3-quals -o Indx90 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx90.txt
tophat -p 10 --solexa1.3-quals -o Indx91 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx91.txt
tophat -p 10 --solexa1.3-quals -o Indx92 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx92.txt
tophat -p 10 --solexa1.3-quals -o Indx93 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx93.txt
tophat -p 10 --solexa1.3-quals -o Indx94 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx94.txt
tophat -p 10 --solexa1.3-quals -o Indx95 -G ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/RNAseq/iGenome/UCSC_hg19/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../Indx_seq/Indx95.txt

Stat.[edit]

  • Mapping rate is lower than previous RNAseq library (60-70%)
  • Samples Indx95 and 96 has severe contamination of mouse genome
Hg19 bowtie.log Indx88 Indx89 Indx90 Indx91 Indx92 Indx93 Indx94 Indx95 Indx96
processed 10,823,774 12,545,719 16,917,879 16,739,939 14,439,641 12,441,496 17,775,293 15,850,762 4,850,658
one alignment 6,030,520 7,206,915 9,344,109 9,210,627 8,199,746 7,211,466 8,315,444 6,324,495 660,512
55.72% 57.45% 55.23% 55.02% 56.79% 57.96% 46.78% 39.90% 13.62%
failed 4,761,793 5,293,227 7,540,575 7,511,226 6,224,057 5,203,180 9,424,809 9,498,774 4,187,420
43.99% 42.19% 44.57% 44.87% 43.10% 41.82% 53.02% 59.93% 86.33%
suppressed -m 31,461 45,577 33,195 18,086 15,838 26,850 35,040 27,493 2,726
0.29% 0.36% 0.20% 0.11% 0.11% 0.22% 0.20% 0.17% 0.06%
Mm9 processed 10,823,774 12,545,719 16,917,879 16,739,939 14,439,641 12,441,496 17,775,293 15,850,762 4,850,658
one alignment 83,419 97,933 126,687 126,258 114,051 88,122 1,442,690 3,232,448 2,099,460
0.77% 0.78% 0.75% 0.75% 0.79% 0.71% 8.12% 20.39% 43.28%
failed 10,738,536 12,445,311 16,788,581 16,611,039 14,322,796 12,351,258 16,322,399 12,591,421 2,734,829
99.21% 99.20% 99.24% 99.23% 99.19% 99.27% 91.83% 79.44% 56.38%
suppressed -m 1,819 2,475 2,611 2,642 2,794 2,116 10,204 26,893 16,369
0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.06% 0.17% 0.34%

Cuffdiff_11.22.11[edit]

  • comparison for Wei: [2]
  • uncertainty about the #4 and #5, b/c separate pair-wise comparisons showed different results/DE gene # from combined all three in one cuffdiff
  1. Indx88:91 - Ctrl/0d vs Ctrl/2d
  2. Indx89:92 - B1/0d vs B1/2d
  3. Indx90:93 - B3/0d vs B3/2d
  4. Indx88:89:90 - Ctrl/0d vs B1/0d vs B3/0d
  5. Indx91:92:93 - Ctrl/2d vs B1/2d vs B3/2d
  • comparison for Dr. Xu
  1. Indx94:95 - Ctrl vs RFP
  2. Indx94:96 - Ctrl vs RFP/GFP
  3. Indx95:96 - RFP vs GFP/RFP

Tophat_11.28.11[edit]

  • Re-run tophat against Ensemble Human dataset

Cuffdiff_11.28.11[edit]

  • Re-run cuffdiff against Ensemble Human dataset