Question: ChIPQC on samples without peaks and wrong total reads
1
gravatar for Amitha Sampath
3.2 years ago by
India
Amitha Sampath10 wrote:

I am trying to use ChIPQC on a set of bam files only (without peaks). I am using the version ChIPQC 1.8.3. So, I created a sample sheet with the bam file and no peak file. I am currently facing 2 issues in generating a ChIPQCreport.

1. I am unable to generate a html report for the experiment (error pasted below)

2. Individual bam file report works fine, but the total number of reads reported are almost double of what is being reported by samtools flagstats or FASTQC (please can you explain why this is happening? FASTQC reports total reads in the fastq file)

Sample sheet 

  SampleID Factor Replicate                    bamReads ControlID                bamControl Peaks PeakCaller
1    Rep_1    GAF         1 R16C_3B-1_C2C12-GAGA_R1.bam     Input R16C_3B-4_C2C12-In_R1.bam    NA        raw
2    Rep_2    GAF         2 R16C_3B-2_C2C12-GAGA_R1.bam     Input R16C_3B-4_C2C12-In_R1.bam    NA        raw
3    Rep_3    GAF         3 R16C_3B-3_C2C12-GAGA_R1.bam     Input R16C_3B-4_C2C12-In_R1.bam    NA        raw

ChIPQC on the above sample sheet

> try_qc = ChIPQC(try,annotation="mm10")
Rep_1  GAF   1 raw
Rep_2  GAF   2 raw
Rep_3  GAF   3 raw
Compiling annotation...
Computing metrics for 4 samples...
list
Bam file has 22 contigs
Calculating coverage histogram for chr10

Calculating SSD for chr10

Calculating shift for chr10

 300 / 300
Counting reads in features for chr10

Calculating coverage histogram for chr11

Calculating SSD for chr11

Calculating shift for chr11

 300 / 300
Counting reads in features for chr11

Calculating coverage histogram for chr12

Calculating SSD for chr12

Calculating shift for chr12

 138 / 300list
Bam file has 22 contigs
Calculating coverage histogram for chr10

Calculating SSD for chr10

Calculating shift for chr10

 300 / 300
Counting reads in features for chr10

Calculating coverage histogram for chr11

Calculating SSD for chr11

Calculating shift for chr11

 300 / 300
Counting reads in features for chr11

Calculating coverage histogram for chr12

Calculating SSD for chr12

Calculating shift for chr12

 138 / 300list
Bam file has 22 contigs
Calculating coverage histogram for chr10

Calculating SSD for chr10

Calculating shift for chr10

 300 / 300
Counting reads in features for chr10

Calculating coverage histogram for chr11

Calculating SSD for chr11

Calculating shift for chr11

 300 / 300
Counting reads in features for chr11

Calculating coverage histogram for chr12

Calculating SSD for chr12

Calculating shift for chr12

 138 / 300list
Bam file has 22 contigs
Calculating coverage histogram for chr10

Calculating SSD for chr10

Calculating shift for chr10

 300 / 300
Counting reads in features for chr10

Calculating coverage histogram for chr11

Calculating SSD for chr11

Calculating shift for chr11

 300 / 300
Counting reads in features for chr11

Calculating coverage histogram for chr12

Calculating SSD for chr12

Calculating shift for chr12

 138 / 300Warning message:
In ChIPQC(try, annotation = "mm10") :
  No chromosomes specified in peaks, using all.

ChIPQCreport 

> ChIPQCreport(try_qc)
Saving 10.1 x 7.46 in image
Saving 10.1 x 7.46 in image
Error in plotCorHeatmap(object, attributes = c(facetBy, colourBy)) : 
  No peaks to plot.

 

Thank you

chipqc • 591 views
ADD COMMENTlink modified 3.2 years ago by Thomas Carroll400 • written 3.2 years ago by Amitha Sampath10
Answer: ChIPQC on samples without peaks and wrong total reads
1
gravatar for Thomas Carroll
3.2 years ago by
United States/New York/The Rockefeller University
Thomas Carroll400 wrote:
hi Amy, The double count for the total reads was due to a duplicated line in earlier ChIPQC Bioconductor 3.3 releases. I fixed this yesterday in the latest version 1.8.6 which should be available today or tomorrow (or is available from SVN in Bioc now). The warning message you see. In ChIPQC(try, annotation = "mm10") : No chromosomes specified in peaks, using all. Come from ChIPQCexperiment using all chromosomes in BAM for the analysis as opposed to selecting from chromosomes in peak files (which it tries when peaks are specified.). This isnt a problem. The error which appears to stop the report is coming from one of the DiffBind plots in ChIPQC, plotCorHeatmap, which hopefully Rory can address. It simply throws an error when no peaks are availalble. If Rory doesnt find a solution i will work around the plot and get back to you when the working version is in Bioconductor repositories. best, tom On Wed, Sep 14, 2016 at 8:58 AM, Amy [bioc] <noreply@bioconductor.org> wrote: > Activity on a post you are following on support.bioconductor.org > > User Amy <https: support.bioconductor.org="" u="" 10057=""/> wrote Question: > ChIPQC on samples without peaks and total reads > <https: support.bioconductor.org="" p="" 87071=""/>: > > I am trying to use ChIPQC on a set of bam files only (without peaks). I am > using the version ChIPQC 1.8.3. So, I created a sample sheet with the bam > file and no peak file. I am currently facing 2 issues in generating a > ChIPQCreport. > > 1. I am unable to generate a html report for the experiment (error pasted > below) > > 2. Individual bam file report works fine, but the total number of reads > reported are almost double of what is being reported by samtools flagstats > or FASTQC (please can you explain why this is happening? FASTQC reports > total reads in the fastq file) > > *Sample sheet * > > SampleID Factor Replicate bamReads ControlID bamControl Peaks PeakCaller > 1 Rep_1 GAF 1 R16C_3B-1_C2C12-GAGA_R1.bam Input R16C_3B-4_C2C12-In_R1.bam NA raw > 2 Rep_2 GAF 2 R16C_3B-2_C2C12-GAGA_R1.bam Input R16C_3B-4_C2C12-In_R1.bam NA raw > 3 Rep_3 GAF 3 R16C_3B-3_C2C12-GAGA_R1.bam Input R16C_3B-4_C2C12-In_R1.bam NA raw > > > *ChIPQC on the above sample sheet* > > > try_qc = ChIPQC(try,annotation="mm10") > Rep_1 GAF 1 raw > Rep_2 GAF 2 raw > Rep_3 GAF 3 raw > Compiling annotation... > Computing metrics for 4 samples... > list > Bam file has 22 contigs > Calculating coverage histogram for chr10 > > Calculating SSD for chr10 > > Calculating shift for chr10 > > 300 / 300 > Counting reads in features for chr10 > > Calculating coverage histogram for chr11 > > Calculating SSD for chr11 > > Calculating shift for chr11 > > 300 / 300 > Counting reads in features for chr11 > > Calculating coverage histogram for chr12 > > Calculating SSD for chr12 > > Calculating shift for chr12 > > 138 / 300list > Bam file has 22 contigs > Calculating coverage histogram for chr10 > > Calculating SSD for chr10 > > Calculating shift for chr10 > > 300 / 300 > Counting reads in features for chr10 > > Calculating coverage histogram for chr11 > > Calculating SSD for chr11 > > Calculating shift for chr11 > > 300 / 300 > Counting reads in features for chr11 > > Calculating coverage histogram for chr12 > > Calculating SSD for chr12 > > Calculating shift for chr12 > > 138 / 300list > Bam file has 22 contigs > Calculating coverage histogram for chr10 > > Calculating SSD for chr10 > > Calculating shift for chr10 > > 300 / 300 > Counting reads in features for chr10 > > Calculating coverage histogram for chr11 > > Calculating SSD for chr11 > > Calculating shift for chr11 > > 300 / 300 > Counting reads in features for chr11 > > Calculating coverage histogram for chr12 > > Calculating SSD for chr12 > > Calculating shift for chr12 > > 138 / 300list > Bam file has 22 contigs > Calculating coverage histogram for chr10 > > Calculating SSD for chr10 > > Calculating shift for chr10 > > 300 / 300 > Counting reads in features for chr10 > > Calculating coverage histogram for chr11 > > Calculating SSD for chr11 > > Calculating shift for chr11 > > 300 / 300 > Counting reads in features for chr11 > > Calculating coverage histogram for chr12 > > Calculating SSD for chr12 > > Calculating shift for chr12 > > 138 / 300Warning message: > In ChIPQC(try, annotation = "mm10") : > No chromosomes specified in peaks, using all. > > *ChIPQCreport * > > > > > ChIPQCreport(try_qc) > Saving 10.1 x 7.46 in image > Saving 10.1 x 7.46 in imageError in plotCorHeatmap(object, attributes = c(facetBy, colourBy)) : > No peaks to plot. > > > > Thank you > > ------------------------------ > > Post tags: chipqc > > You may reply via email or visit ChIPQC on samples without peaks and wrong total reads >
ADD COMMENTlink written 3.2 years ago by Thomas Carroll400

Thanks Tom! I will try with the new version in that case :-) 

ADD REPLYlink written 3.2 years ago by Amitha Sampath10
1

If there are no peaks, we should still generate a report and simply skip the plots that only make sense if there are peaks, rather than report an error. We are already testing for this condition and reporting an error, we just need to change the test that prints out the "No peaks to plot" lines to something that returns cleanly. 

I'll coordinate an appropriate response with Tom.

-Rory

ADD REPLYlink written 3.2 years ago by Rory Stark2.9k
Answer: ChIPQC on samples without peaks and wrong total reads
0
gravatar for Thomas Carroll
3.2 years ago by
United States/New York/The Rockefeller University
Thomas Carroll400 wrote:

hi Amitha,

I have added a fix to skip the troublesome plots in ChIPQC version 1.8.8.

best,

tom.

ADD COMMENTlink written 3.2 years ago by Thomas Carroll400
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