Rui:Data Analysis: Difference between revisions

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==Examples==
==Examples==
KUN's pipeline:
http://genome-tech.ucsd.edu/LabNotes/index.php/RNAseq_pipeline [http://genome-tech.ucsd.edu/LabNotes/index.php/RNAseq_pipeline]
  Jeff's samples:
  Jeff's samples:
  Kun:LabNotes/Haplotyping/2010-9-17 [http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/Haplotyping/2010-9-17]
  Kun:LabNotes/Haplotyping/2010-9-17 [http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/Haplotyping/2010-9-17]
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*[[Rui:Haplotyping 6.21.11|Haplotyping 6.21.11]]
*[[Rui:Haplotyping 6.21.11|Haplotyping 6.21.11]]
*[[Rui:RNAseq analysis from 7.18.11|RNAseq analysis from 7.18.11]]
*[[Rui:RNAseq analysis from 7.18.11|RNAseq analysis from 7.18.11]]
*[[Rui:RNAseq analysis on HL098lRNAseq analysis on HL098]]
*[[Rui:RNAseq analysis on HL098|RNAseq analysis on HL098]]
*[[Rui:RNAseq analysis on HL099|RNAseq analysis on HL099]]
*[[Rui:RNAseq analysis on HL099|RNAseq analysis on HL099]]
*[[Rui:RNAseq analysis on HL101|RNAseq analysis on HL101]]
*[[Rui:RNAseq analysis on Hiseq111005|RNAseq_PGCsamples on Hiseq111005]]
*[[Rui:Hap analysis on HL108|Hap analysis on HL108]]
*[[Rui:RNAseq analysis on HL109|RNAseq analysis on HL109]]
*[[Rui:DNAseq analysis on HL114|DNAseq analysis on HL114]]
*[[Rui:DNAseq analysis on Hiseq120313|DNAseq analysis on Hiseq120313]]
*[[Rui:DNAseq analysis on HL118|DNAseq analysis on HL118]]
*[[Rui:totoRNAseq on 121226_HL140|totoRNAseq on 121226_HL140]]
*[[Rui:totoRNAseq on 130120_HL141|totoRNAseq on 130120_HL141]]
*[[Rui:totoRNAseq on 130220_HL144|totoRNAseq on 130220_HL144]]
*[[Rui:totoRNAseq on BL-UHRR-IVT-May28_2013|totoRNAseq on BL-UHRR-IVT-May28_2013]]
*[[Rui:totoRNAseq on 131009_HL161|totoRNAseq on 131009_HL161]]


==RNAseq analysis on HL098==
==RNAseq analysis key notes==
===Database download===
* iGenome packages [http://tophat.cbcb.umd.edu/igenomes.html]
iGenome collection [http://cufflinks.cbcb.umd.edu/igenomes.html], save in ~/iGenome/
# UCSC genome package: [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:Tophat_mapping_10.27.11]
 
# Ensemble genome package: [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:Tophat_11.21.11#Re-download_Ensemble_package_from_iGenome_.5B1.5D]
* wget ftp://igenome:G3nom3s4u@ftp.illumina.com/Homo_sapiens/UCSC/hg19/Homo_sapiens_UCSC_hg19.tar.gz
* wget ftp://igenome:G3nom3s4u@ftp.illumina.com/Mus_musculus/UCSC/mm9/Mus_musculus_UCSC_mm9.tar.gz
* tar -zxvf Homo_sapiens_UCSC_hg19.tar.gz
* tar -zxvf Mus_musculus_UCSC_mm9.tar.gz
 
* BowtieIndex: ~/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex | ~/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex
* Annotation: ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf
 
===Tophat===
nohup tophat -p 6 --solexa1.3-quals -o ./tophat-g/ -r 250 --mate-std-dev 80 -G ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ./seq/s_1_1_Indx1.txt ./seq/s_1_2_Indx1.mod.txt
 
nohup tophat -p 6 --solexa1.3-quals -o ./tophat-g/ -r 250 --mate-std-dev 80 -G ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf ~/iGenome/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ./seq/s_1_1_Indx2.txt ./seq/s_1_2_Indx2.mod.txt
 
===Samtools and clonalreads removal===
samtools flagstat accepted_hits.bam
 
~/RNAtools/removalclonalreads.pl accepted_hits.bam
 
 
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''Pair-end mapping'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Tophat'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''removeClonalHits.pl'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||||file size||fastq reads in||reads out||%||options||accepted hits||reads hits||pair/single||Properly paired||% of total||mapped reads||% of total||pro-rm clonal reads||% of total||% of mappable reads||then uniquely mapped||% of total||potential coverage
|-
| s_1_Indx1||s_1_1_Indx1||left read||2,307,029||2,300,738||0.9973||r:250||1,705,993||734,602||1,122,540||||0.657998011||1,615,384||0.3501||1,191,029||0.2581||0.7373||1,160,079||0.2514||0.04640316
|-
| ||s_1_2_Indx1||right read||2,307,029||2,304,795||0.9990||STD:80||||971,391||583,453||941,458||0.551853378||||||||||||||||
|-
| s_1_Indx1||s_1_1_Indx1||left read||2,307,029||2,300,738||0.9973||(+g)||1,733,904||746,989||1,156,628||||0.667065766||1,643,007||0.3561||1,218,033||0.2640||0.7413||1,186,899||0.2572||0.04747596
|-
| ||s_1_2_Indx1||right read||2,307,029||2,304,795||0.9990||UCSC||||986,915||577,276||965,638||0.556915492||||||||||||||||
|-
| ||||||||||||||||||||||||||||||||||||||
|-
| s_1_Indx2||s_1_1_Indx2||left read||3,078,335||3,069,878||0.9973||r:250||3,882,019||1,679,990||2,750,090||||0.70841745||3,678,987||0.5976||2,642,026||0.4291||0.7181||2,585,255||0.4199||0.1034102
|-
| ||s_1_2_Indx2||right read||3,078,335||3,075,318||0.9990||STD:80||||2,202,029||1,131,929||2,293,768||0.590869854||||||||||||||||
|-
| s_1_Indx2||s_1_1_Indx2||left read||3,078,335||3,069,878||0.9973||(+g)||3,913,815||1,694,018||2,792,224||||0.713427691||3,711,732||0.6029||2,674,065||0.4343||0.7204||2,617,136||0.4251||0.10468544
|-
| ||s_1_2_Indx2||right read||3,078,335||3,075,318||0.9990||UCSC||||2,219,797||1,121,591||2,344,834||0.59911723||||||||||||||||
|}
 
===Cufflinks and cuffdiff===
samtools view -h ../accepted_hits.bam > accepted_hits.sam (have to convert to sam first, i can't feed cufflinks with bam file --- error: segment fault...)


cufflinks -g ~/iGenome/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf -m 250 -s 80 accepted_hits.sam (-g option is probably unnecessary...)
* Comparison of RNAseq analysis programs: [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:RNAseq_analysis_on_Hiseq111005#Comparison_of_program]

Latest revision as of 21:02, 31 October 2013

Examples[edit]

KUN's pipeline:
http://genome-tech.ucsd.edu/LabNotes/index.php/RNAseq_pipeline [1]
Jeff's samples:
Kun:LabNotes/Haplotyping/2010-9-17 [2]
Transcriptome:
Kun:LabNotes/SingleCellExpr/2011-6-13 [3]
Haplotyping:
Kun:LabNotes/Haplotyping/2011-6-14 [4]

Data analysis[edit]

RNAseq analysis key notes[edit]

  • iGenome packages [5]
  1. UCSC genome package: [6]
  2. Ensemble genome package: [7]
  • Comparison of RNAseq analysis programs: [8]