Kun:LabNotes/SingleCellExpr/2011-9-7
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RNAseq data analysis
- The data came from limited number of mouse PGC cells provided by Yi Zhang's lab. Rui made the RNA sequencing libraries.
Analysis of low-pass sequencing data.
- The first round of low-pass sequencing was done in HL101 run (SE60bp+index), lane 7-8.
- I ran tophat on each data set, using mouse gene annotation as the model.
less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx3.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx3.txt.gz > RNAseq_Indx3.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx4.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx4.txt.gz > RNAseq_Indx4.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx5.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx5.txt.gz > RNAseq_Indx5.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx6.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx6.txt.gz > RNAseq_Indx6.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx7.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx7.txt.gz > RNAseq_Indx7.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx8.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx8.txt.gz > RNAseq_Indx8.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx9.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx9.txt.gz > RNAseq_Indx9.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx10.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx10.txt.gz > RNAseq_Indx10.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx11.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx11.txt.gz > RNAseq_Indx11.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx12.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx12.txt.gz > RNAseq_Indx12.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx13.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx13.txt.gz > RNAseq_Indx13.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx14.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx14.txt.gz > RNAseq_Indx14.fq less /home/kunzhang/SeqStore/110902_HL101/s_7_Indx15.txt.gz /home/kunzhang/SeqStore/110902_HL101/s_8_Indx15.txt.gz > RNAseq_Indx15.fq /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 RNAseq_Indx3.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx4 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx4.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx5 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx5.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx6 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx6.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx7 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx7.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx8 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx8.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx9 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx9.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx10 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx10.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx11 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx11.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx12 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx12.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx13 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx13.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx14 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx14.fq /home/kunzhang/softwares/tophat-latest/tophat -p 8 --solexa1.3-quals -o tophat_Mm_Indx15 -G /GenomeDB/UCSC/Mus_musculus/mm9/Annotation/Genes/genes.gtf /GenomeDB/UCSC/Mus_musculus/mm9/Sequence/BowtieIndex/genome RNAseq_Indx15.fq
- Then I checked the tophat_Mm_IndxN/logs/bowtie.left_kept_reads.fixmap.log files, and used the mapping rates in these files as the proxy of the true mapping rates.
Sample Name | Index | Qualty-filtered reads | Uniquely mappable reads | Non-mappable reads | Mapping rate |
E9.5 | Indx3 | 3,775,311 | 2,715,872 | 1,014,808 | 71.9% |
E11.5 | Indx4 | 3,987,505 | 2,710,512 | 1,238,018 | 68.0% |
wt E13.5m1 | Indx5 | 4,212,162 | 2,921,411 | 1,250,730 | 69.4% |
wt E13.5f1 | Indx6 | 3,557,137 | 2,506,010 | 1,019,618 | 70.5% |
E9.5 | Indx7 | 5,628,397 | 3,964,069 | 1,571,087 | 70.4% |
E11.5 | Indx8 | 6,095,896 | 4,322,793 | 1,687,193 | 70.9% |
wt E13.5m1 | Indx9 | 6,284,443 | 4,290,999 | 1,925,123 | 68.3% |
wt E13.5m2 | Indx10 | 4,649,460 | 3,276,710 | 1,316,390 | 70.5% |
wt E13.5f1 | Indx11 | 5,953,433 | 4,199,249 | 1,693,646 | 70.5% |
wt E13.5f2 | Indx12 | 6,712,315 | 4,671,588 | 1,969,301 | 69.6% |
KO E13.5m | Indx13 | 4,954,624 | 3,517,633 | 1,380,709 | 71.0% |
KO E13.5f1 | Indx14 | 7,223,059 | 5,137,419 | 1,999,302 | 71.1% |
KO E13.5f2 | Indx15 | 8,053,410 | 5,681,254 | 2,272,034 | 70.5% |
- Conclusions:
- The mapping rate is consistently at ~70% across all libraries.
- To generate roughly 20 millions mappable reads in each library, we need 4-5 HiSeq sequencing lanes.
Further examination of the data quality
- I first create a master transcripts.gtf file that combined all transcripts found in the 13 data sets.
/home/kunzhang/softwares/cufflinks-1.0.3.Linux_x86_64/cuffcompare -o cuffcompare_all -R tophat_Mm_Indx3/transcripts.gtf tophat_Mm_Indx4/transcripts.gtf ... tophat_Mm_Indx15/transcripts.gtf
- Then I ran cuffdiff on technical/biological replicates to examine the noise in the data.
/home/kunzhang/softwares/cufflinks-1.0.3.Linux_x86_64/cuffdiff cuffcompare_all.combined.gtf -N -p 8 -o cuffDiff_3_vs_7 tophat_Mm_Indx3/accepted_hits.bam tophat_Mm_Indx7/accepted_hits.bam /home/kunzhang/softwares/cufflinks-1.0.3.Linux_x86_64/cuffdiff cuffcompare_all.combined.gtf -N -p 8 -o cuffDiff_4_vs_8 tophat_Mm_Indx4/accepted_hits.bam tophat_Mm_Indx8/accepted_hits.bam /home/kunzhang/softwares/cufflinks-1.0.3.Linux_x86_64/cuffdiff cuffcompare_all.combined.gtf -N -p 8 -o cuffDiff_9_vs_10 tophat_Mm_Indx9/accepted_hits.bam tophat_Mm_Indx10/accepted_hits.bam /home/kunzhang/softwares/cufflinks-1.0.3.Linux_x86_64/cuffdiff cuffcompare_all.combined.gtf -N -p 8 -o cuffDiff_14_vs_15 tophat_Mm_Indx14/accepted_hits.bam tophat_Mm_Indx15/accepted_hits.bam
- Then I plotted the FPKM values between each pairs of replicates.
File:FPKM Indx3 vs Indx7.pngFile:FPKM Indx4 vs Indx8.png File:FPKM Indx9 vs Indx10.pngFile:FPKM Indx14 vs Indx15.png