Matt:LabNotes/2014-10-31
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RT Primer RNA-Seq Analysis
- Library Matt:LabNotes/2014-9-25
Align with Tophat2 to hg19
~/scratch/RTprimer_Analysis$ /home/kunzhang/softwares/tophat-2.0.6.Linux_x86_64/tophat2 -o tophat_hg19unmask_Indx26_RanHex --solexa1.3-quals ~/LTS/Genomes/hg19 s_8_1_Indx26.txt ~/scratch/RTprimer_Analysis$ /home/kunzhang/softwares/tophat-2.0.6.Linux_x86_64/tophat2 -o tophat_hg19unmask_Indx27_dT --solexa1.3-quals ~/LTS/Genomes/hg19 s_8_1_Indx27.txt ~/scratch/RTprimer_Analysis$ /home/kunzhang/softwares/tophat-2.0.6.Linux_x86_64/tophat2 -o tophat_hg19unmask_Indx28_FISSEQRT --solexa1.3-quals ~/LTS/Genomes/hg19 s_8_1_Indx28.txt ~/scratch/RTprimer_Analysis$ /home/kunzhang/softwares/tophat-2.0.6.Linux_x86_64/tophat2 -o tophat_hg19unmask_Indx29_Top48 --solexa1.3-quals ~/LTS/Genomes/hg19 s_8_1_Indx29.txt
- RanHex
586553 reads; of these: 586553 (100.00%) were unpaired; of these: 251516 (42.88%) aligned 0 times 196622 (33.52%) aligned exactly 1 time 138415 (23.60%) aligned >1 times 57.12% overall alignment rate
- dT
432896 reads; of these: 432896 (100.00%) were unpaired; of these: 103736 (23.96%) aligned 0 times 215595 (49.80%) aligned exactly 1 time 113565 (26.23%) aligned >1 times 76.04% overall alignment rate
- FISSEQRT
529281 reads; of these: 529281 (100.00%) were unpaired; of these: 199427 (37.68%) aligned 0 times 205336 (38.80%) aligned exactly 1 time 124518 (23.53%) aligned >1 times 62.32% overall alignment rate
- Top48
523107 reads; of these: 523107 (100.00%) were unpaired; of these: 165872 (31.71%) aligned 0 times 229706 (43.91%) aligned exactly 1 time 127529 (24.38%) aligned >1 times 68.29% overall alignment rate
Samtools Sort and Index
samtools sort tophat_hg19unmask_Indx26_RanHex/accepted_hits.bam mapped_RanHex.sorted samtools sort tophat_hg19unmask_Indx27_dT/accepted_hits.bam mapped_dT.sorted samtools sort tophat_hg19unmask_Indx28_FISSEQRT/accepted_hits.bam mapped_FISSEQRT.sorted samtools sort tophat_hg19unmask_Indx29_Top48/accepted_hits.bam mapped_Top48.sorted
samtools index mapped_RanHex.sorted.bam mapped_RanHex.sorted.bam.bai samtools index mapped_dT.sorted.bam mapped_dT.sorted.bam.bai samtools index mapped_FISSEQRT.sorted.bam mapped_FISSEQRT.sorted.bam.bai samtools index mapped_Top48.sorted.bam mapped_Top48.sorted.bam.bai
fetchChromSizes hg19 > ~/Genomes/hg19.chrom.sizes
Visual QC
bam2wig.py -i mapped_RanHex.sorted.bam -s /home/mzcai/LTS/Genomes/hg19.chrom.sizes -o mapped_RanHex.sorted bam2wig.py -i mapped_dT.sorted.bam -s /home/mzcai/LTS/Genomes/hg19.chrom.sizes -o mapped_dT.sorted bam2wig.py -i mapped_FISSEQRT.sorted.bam -s /home/mzcai/LTS/Genomes/hg19.chrom.sizes -o mapped_FISSEQRT.sorted bam2wig.py -i mapped_Top48.sorted.bam -s /home/mzcai/LTS/Genomes/hg19.chrom.sizes -o mapped_Top48.sorted
Calculate rRNA Overlap
hg19_rRNA.bed from UCSC table browser
split_bam.py -i mapped_RanHex.sorted.bam -r /home/mzcai/LTS/Genomes/hg19_rRNA.bed -o split_hg19rRNAbed_RanHex Total records: 398868 split_hg19rRNAbed_RanHex.in.bam (Reads consumed by input gene list):251593 split_hg19rRNAbed_RanHex.ex.bam (Reads not consumed by input gene list):147275 split_hg19rRNAbed_RanHex.junk.bam (qcfailed, unmapped reads):0
split_bam.py -i mapped_dT.sorted.bam -r /home/mzcai/LTS/Genomes/hg19_rRNA.bed -o split_hg19rRNAbed_dT Total records: 422589 split_hg19rRNAbed_dT.in.bam (Reads consumed by input gene list):155754 split_hg19rRNAbed_dT.ex.bam (Reads not consumed by input gene list):266835 split_hg19rRNAbed_dT.junk.bam (qcfailed, unmapped reads):0
split_bam.py -i mapped_FISSEQRT.sorted.bam -r /home/mzcai/LTS/Genomes/hg19_rRNA.bed -o split_hg19rRNAbed_FISSEQRT Total records: 391847 split_hg19rRNAbed_FISSEQRT.in.bam (Reads consumed by input gene list):220685 split_hg19rRNAbed_FISSEQRT.ex.bam (Reads not consumed by input gene list):171162 split_hg19rRNAbed_FISSEQRT.junk.bam (qcfailed, unmapped reads):0
split_bam.py -i mapped_Top48.sorted.bam -r /home/mzcai/LTS/Genomes/hg19_rRNA.bed -o split_hg19rRNAbed_Top48 Total records: 422471 split_hg19rRNAbed_Top48.in.bam (Reads consumed by input gene list):237568 split_hg19rRNAbed_Top48.ex.bam (Reads not consumed by input gene list):184903 split_hg19rRNAbed_Top48.junk.bam (qcfailed, unmapped reads):0
Homo_sapiens.GRCh37.75.totalrRNA.chr.bed
- Bed file from gene annotations of Hg19 from Ensembl
- Converted gtf to bed
- Added 'chr' to chromosome names
split_bam.py -i mapped_RanHex.sorted.bam -r /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.totalrRNA.chr.bed -o split_GRCh37totalrRNAchrbed_RanHex Total records: 398868 split_GRCh37totalrRNAchrbed_RanHex.in.bam (Reads consumed by input gene list):47 split_GRCh37totalrRNAchrbed_RanHex.ex.bam (Reads not consumed by input gene list):398821 split_GRCh37totalrRNAchrbed_RanHex.junk.bam (qcfailed, unmapped reads):0
split_bam.py -i mapped_dT.sorted.bam -r /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.totalrRNA.chr.bed -o split_GRCh37totalrRNAchrbed_dT Total records: 422589 split_GRCh37totalrRNAchrbed_dT.in.bam (Reads consumed by input gene list):13 split_GRCh37totalrRNAchrbed_dT.ex.bam (Reads not consumed by input gene list):422576 split_GRCh37totalrRNAchrbed_dT.junk.bam (qcfailed, unmapped reads):0
split_bam.py -i mapped_FISSEQRT.sorted.bam -r /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.totalrRNA.chr.bed -o split_GRCh37totalrRNAchrbed_FISSEQRT Total records: 391847 split_GRCh37totalrRNAchrbed_FISSEQRT.in.bam (Reads consumed by input gene list):43 split_GRCh37totalrRNAchrbed_FISSEQRT.ex.bam (Reads not consumed by input gene list):391804 split_GRCh37totalrRNAchrbed_FISSEQRT.junk.bam (qcfailed, unmapped reads):0
split_bam.py -i mapped_Top48.sorted.bam -r /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.totalrRNA.chr.bed -o split_GRCh37totalrRNAchrbed_Top48 Total records: 422471 split_GRCh37totalrRNAchrbed_Top48.in.bam (Reads consumed by input gene list):16 split_GRCh37totalrRNAchrbed_Top48.ex.bam (Reads not consumed by input gene list):422455 split_GRCh37totalrRNAchrbed_Top48.junk.bam (qcfailed, unmapped reads):0
Map with Tophat2
- --report-secondary-alignments