Question: Chipseq peak analysis using ChIPseeker
0
gravatar for Mike
3.3 years ago by
Mike1.4k
UK
Mike1.4k wrote:

Hi ,

I am using ChIPseeker tool for my Chipseq data analysis, But my heatmap of ChiP binding to TSS regions is almost blank “attached image”, enter image description here

I used macs2 output file.

I used following commands:

txdb <- TxDb.Hsapiens.UCSC.hg19.knownGene


peak <- readPeakFile("chip_vs_input_summits.bed")
covplot(peak, weightCol="V5")



##Profile of ChIP peaks binding to TSS regions
promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
tagMatrix <- getTagMatrix(peak, windows=promoter)
tagHeatmap(tagMatrix, xlim=c(-3000, 3000), color="red")
chipseeker chip-seq genome • 2.9k views
ADD COMMENTlink modified 3.3 years ago by Guangchuang Yu2.3k • written 3.3 years ago by Mike1.4k

Sorry in above case I used peak summit file, now I tried with *peaks.narrowPeak macs2 output files,

awk 'BEGIN {OFS="\t"} {print $1,$2,$3,$4,$7,$9}'  chip_vs_input_peaks.narrowPeak >chip_vs_input.bed

when I used this file it gives some warnings:

 promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
tagMatrix <- getTagMatrix(peak, windows=promoter)
Warning message:
In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:
  - in 'x': chr20, chr21, chr22, chrY, chrM, chr1_gl000191_random, chr1_gl000192_random, chr4_ctg9_hap1, chr4_gl000193_random, chr4_gl000194_random, chr6_apd_hap1, chr6_cox_hap2, chr6_dbb_hap3, chr6_mann_hap4, chr6_mcf_hap5, chr6_qbl_hap6, chr6_ssto_hap7, chr7_gl000195_random, chr8_gl000196_random, chr8_gl000197_random, chr9_gl000198_random, chr9_gl000199_random, chr9_gl000200_random, chr9_gl000201_random, chr11_gl000202_random, chr17_ctg5_hap1, chr17_gl000203_random, chr17_gl000204_random, chr17_gl000205_random, chr17_gl000206_random, chr18_gl000207_random, chr19_gl000208_random, chr19_gl000209_random, chr21_gl000210_random, chrUn_gl000211, chrUn_gl000212, chrUn_gl000213, chrUn_gl000214, chrUn_gl000215, chrUn_gl000216, chrUn_gl000217, chrUn_gl000218, chrUn_gl000219, chrUn_gl000220, chrUn_gl000221, chrUn_gl000222, chrUn_gl000223, chrUn_gl000224, chrUn_gl000225, chrUn_gl000226, chrUn_gl000227, chrUn_gl000228, chrUn_gl0 [... truncated]
ADD REPLYlink modified 3.3 years ago • written 3.3 years ago by Mike1.4k

maybe send me your bed file and source code for me to reproduce the issue.

ADD REPLYlink written 3.3 years ago by Guangchuang Yu2.3k

Thanks,

this is my macs2 command:

macs2 callpeak -t chip.bam -c input.bam -f BAM -B -g hs -n rep1

and input bed file:

http://www.filedropper.com/showdownload.php/sample1peaks

or

http://s000.tinyupload.com/index.php?file_id=66463745037323321210

ADD REPLYlink modified 3.3 years ago • written 3.3 years ago by Mike1.4k
1
gravatar for Guangchuang Yu
3.3 years ago by
Guangchuang Yu2.3k
China/Guangzhou/Southern Medical University
Guangchuang Yu2.3k wrote:
Warning message:
In .Seqinfo.mergexy(x, y) :
  Each of the 2 combined objects has sequence levels not in the other:

The warning message is OK, as you can check:

> seqlevels(txdb)
 [1] "chr1"                  "chr2"                  "chr3"
 [4] "chr4"                  "chr5"                  "chr6"
 [7] "chr7"                  "chr8"                  "chr9"
[10] "chr10"                 "chr11"                 "chr12"
[13] "chr13"                 "chr14"                 "chr15"
[16] "chr16"                 "chr17"                 "chr18"
[19] "chr19"                 "chr20"                 "chr21"
[22] "chr22"                 "chrX"                  "chrY"
[25] "chrM"                  "chr1_gl000191_random"  "chr1_gl000192_random"
[28] "chr4_ctg9_hap1"        "chr4_gl000193_random"  "chr4_gl000194_random"
[31] "chr6_apd_hap1"         "chr6_cox_hap2"         "chr6_dbb_hap3"
[34] "chr6_mann_hap4"        "chr6_mcf_hap5"         "chr6_qbl_hap6"
[37] "chr6_ssto_hap7"        "chr7_gl000195_random"  "chr8_gl000196_random"
[40] "chr8_gl000197_random"  "chr9_gl000198_random"  "chr9_gl000199_random"
[43] "chr9_gl000200_random"  "chr9_gl000201_random"  "chr11_gl000202_random"
[46] "chr17_ctg5_hap1"       "chr17_gl000203_random" "chr17_gl000204_random"
[49] "chr17_gl000205_random" "chr17_gl000206_random" "chr18_gl000207_random"
[52] "chr19_gl000208_random" "chr19_gl000209_random" "chr21_gl000210_random"
[55] "chrUn_gl000211"        "chrUn_gl000212"        "chrUn_gl000213"
[58] "chrUn_gl000214"        "chrUn_gl000215"        "chrUn_gl000216"
[61] "chrUn_gl000217"        "chrUn_gl000218"        "chrUn_gl000219"
[64] "chrUn_gl000220"        "chrUn_gl000221"        "chrUn_gl000222"
[67] "chrUn_gl000223"        "chrUn_gl000224"        "chrUn_gl000225"
[70] "chrUn_gl000226"        "chrUn_gl000227"        "chrUn_gl000228"
[73] "chrUn_gl000229"        "chrUn_gl000230"        "chrUn_gl000231"
[76] "chrUn_gl000232"        "chrUn_gl000233"        "chrUn_gl000234"
[79] "chrUn_gl000235"        "chrUn_gl000236"        "chrUn_gl000237"
[82] "chrUn_gl000238"        "chrUn_gl000239"        "chrUn_gl000240"
[85] "chrUn_gl000241"        "chrUn_gl000242"        "chrUn_gl000243"
[88] "chrUn_gl000244"        "chrUn_gl000245"        "chrUn_gl000246"
[91] "chrUn_gl000247"        "chrUn_gl000248"        "chrUn_gl000249"
> seqlevels(peak)
 [1] "chr1"         "chr1_random"  "chr10"        "chr11"        "chr12"
 [6] "chr13"        "chr13_random" "chr14"        "chr15"        "chr16"
[11] "chr17"        "chr18"        "chr19"        "chr2"         "chr3"
[16] "chr4"         "chr5"         "chr6"         "chr7"         "chr8"
[21] "chr8_random"  "chr9"         "chrUn_random" "chrX"         "chrY"
[26] "chrY_random"

I can produce the tagHeatmap using the sample bed file you provided.

screenshot 2016-09-07 21 24 24

ADD COMMENTlink written 3.3 years ago by Guangchuang Yu2.3k

Thanks you so much Guangchuang Yu,

what are chr1/13/8_random and chrUn_random, why some chr (chr20, ch21, chr22 ) are missing in my data.

Thanks.

ADD REPLYlink written 3.3 years ago by Mike1.4k

sorry one more problem,

promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)
tagMatrixList <- lapply(files, getTagMatrix, windows=promoter)
plotAvgProf(tagMatrixList, xlim=c(-3000, 3000))

I get this error

Error in plotAvgProf.internal(tagMatrix, xlim = xlim, xlab = xlab, ylab = ylab,  : 
  tagMatrix should be a named list...

how to give list of bed files in plotAvgProf

but below command is works:

 tagHeatmap(tagMatrixList, xlim=c(-3000, 3000), color=NULL)

And when I use :

peakAnnoList <- lapply(files, annotatePeak, TxDb=txdb, tssRegion=c(-3000, 3000), verbose=FALSE)
 plotAnnoBar(peakAnnoList)

error:

Error in eval(expr, envir, enclos) : object '.id' not found
ADD REPLYlink modified 3.3 years ago • written 3.3 years ago by Mike1.4k

just named your files.

names(files) <- basename(files)

or

names(files) <- paste0('file_', seq_along(files))

something like this.

ADD REPLYlink written 3.3 years ago by Guangchuang Yu2.3k

I don't know either.

ADD REPLYlink written 3.3 years ago by Guangchuang Yu2.3k
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