flat plots in ngsplots

[ShivPet]

HI Li,
Need some urgent help. I have the same issue. My code was:
ngs.plot.r -G hg19 -R tss -C P074A2_S5_R1_001.fastq.trim.align.BLMTfil.sort.rmdup.bam -O P074A1.tss -T P074A2TSS. The data is peaks called using MACS2. My plots are like so:

P074A1.tss.heatmap.pdf
P074A1.tss.avgprof.pdf

Kindly help. Im a total novice and have no idea what to do.

Thanks
Shiva.

[amitpande74]

H shivPet,
Can you tel us something more about your BAM file.....how did you process it ....sorting etc and if your data are the peaks from MACS2 then why are you using the -R tss flag ?
You should be using ngs.plot.r -G hg19 -R bed -E your peak file.bed -C bam file -O P074A1.tss -T P074A2TSS.
try it !

[ShivPet]

Hi Amitpande,
Thanks so much for your prompt reply. My data is actually ATAC-seq data.I just wanted to initially see if my reads from my alignment are (slightly) enriched at the TSS sites- which is how it seems visually from IGV. The code for generating and processing my bam files are in the attached pdf
ATAC-Seq pipeline-pdf.pdf.
meanwhile ill try your suggestion using my peak files.

Thanks,
Shiva

[ADSL0067]

Hi, Amitpande
I also get issue as same as ShivPet met. The bam file (GSM733659) was downloaded from ncbi which made by maq, sorted by samtools and remove the duplication by picard. ngs.plot can make the enrichment results using the default TSS which attached by the sofeware, but when use TSS.bed ( format: chr1<tab>91104<tab>91105<tab>ENSG00000239945.1 ) which make by myselfe, it's no enrichment pattern. And the TSS.bed can working perfect with the bam made by bowtie. What should i do?
MY code:
ngs.plot2.r -G hg19 -R bed -E Gencode_CodingANDlincRNA.filter.bed -C GSM733659_hg19_Helas3Control.rmdup.bam -O GSM733659_hg19_Helas3Control -SC 0,1 -GO prod -LOW 0 -L 2000 -FL 100 -T GSM733659 -RR 60 -CO Coral

Any advice is welcome.

Thanks,

Liang

[lishen]

When you see a flat plot, the first thing to check is whether your bam files contain the same chromosome names as your references. If not, you can reheader the bam files.

Also, I just release 2.63 which fixes some critical issues. Be sure to use the new version.

[sinirautio]

Hi,

were you able to solve the problem? I have exactly the same problem, haven't had it previously but now I downloaded the data from NCBI and it just shows a flat plot even though I know there are reads aligning to those regions in the bed file.