Rui:Data Analysis: Difference between revisions

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*[[Rui:RNAseq analysis on HL098|RNAseq 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]]
==RNAseq analysis on HL098==
===Database download===
iGenome collection [http://cufflinks.cbcb.umd.edu/igenomes.html], save in ~/iGenome/
* 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...)

Revision as of 23:19, 14 October 2011

Examples

Jeff's samples:
Kun:LabNotes/Haplotyping/2010-9-17 [1]
Transcriptome:
Kun:LabNotes/SingleCellExpr/2011-6-13 [2]
Haplotyping:
Kun:LabNotes/Haplotyping/2011-6-14 [3]

Data analysis