Matt:LabNotes/2014-10-31: Difference between revisions

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>Mzcai
(Created page with "==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...")
 
>Mzcai
mNo edit summary
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     127529 (24.38%) aligned >1 times
     127529 (24.38%) aligned >1 times
   68.29% overall alignment rate
   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
<!-- wigToBigWig wigVarStepExample.gz hg19.chrom.sizes myBigWig.bw -->
===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 [[Matt:LabNotes/2014-7-14#Bedtools_intersect | gene annotations of Hg19 from Ensembl]]
**Converted gtf to bed
**[[Matt:LabNotes/2014-7-24#Bedtools_intersect_troubleshooting | 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

Revision as of 03:59, 3 November 2014

RT Primer RNA-Seq Analysis

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

 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