Question on TAD parameter settings

[YiChienLee1010]

Hi
I want to plot TADs on my matrix but have some problems and I am not sure which step caused the issue.

I am using Omni-C data, and I create 256 sets of 4 base random restriction enzyme sites to build a matrix.
My commands for each step are as follows.

#mapping
bwa mem -A1 -B4 -E50 -L0 -t 8 assembly.fa LGH24_R1_v2.fastq.gz > LGH24_R1_v2.sam (same for R2)
samtools view -@ 32 -Shb LGH24_R1_v2.sam > LGH24_R2_v2.bam (same for R2)

#hicFindRestSite
cat random_site.txt | parallel -j 5 'hicFindRestSite --fasta ref.fa --searchPattern {} --outFile cutsites{}.bed'
*random_site.txt contain all 256 randome restriction enzyme site 

#hicBuildMatrix
hicBuildMatrix -s LGH24_R1_v2.bam LGH24_R2_v2.bam --outFileName Matrix_bin50000_scaff0001.h5 --QCfolder Matrix_bin50000_scaff0001_QC --restrictionCutFile (list all 256 cutsites) --restrictionSequence (list all 256 randome sequences)  --danglingSequence (list all 256 randome sequences)  -b Matrix_bin50000_scaff0001.bam --binSize 50000 --threads 8  --region scaff0001

#hicCorrectMatrix
hicCorrectMatrix diagnostic_plot --matrix Matrix_bin50000_scaff0001.h5 -o hicCorrectMatrix_bin50000_scaff0001.png
hicCorrectMatrix correct --matrix  Matrix_bin50000_scaff0001.h5 --filterThreshold -1.5 5  -o hicCorrectMatrix_bin50000_scaff0001.h5

#hicFindTADs
hicFindTADs --matrix hicCorrectMatrix_bin50000_scaff0001.h5 --outPrefix hicCorrectMatrix_bin50000_scaff0001_TAD_default --correctForMultipleTesting fdr 
make_tracks_file --trackFiles hicCorrectMatrix_bin50000_scaff0001.h5 hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains.bed -o scaff0001_TAD_default.ini
hicPlotTADs --tracks scaff0001_TAD_default.ini --region scaff0001:1-178105659 -o scaff0001_TAD_default.png

scaff0001_TAD_default.ini

[x-axis]
fontsize = 10

[hicCorrectMatrix_bin50000_scaff0001]
file = hicCorrectMatrix_bin50000_scaff0001.h5
depth = 1000
transform = log1p
file_type = hic_matrix
height = 15
show_masked_bins = false

[hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains]
file = hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains.bed
file_type = domains
border_color = black
color = none
display = triangles
title = hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains
height = 15
overlay_previous = share-y
labels = false
fontsize = 10

scaff0001_TAD_default.png

I also tried --binSize 10000 at the hicBuildMatrix step, but it also doesn't work.
I had set different highs to see any difference, but it still looked weird. I had set depths from 10kb, 40kb, and 120kb, but also no better result.

Is this related to bin size? or the plotting parameter?
Hicexplorer version 3.7.6

Any suggestions?
Thanks!

[joachimwolff]

The area you are plotting is too large for the width you define. Try sth like 5 MB with the current width and see how that goes. Or increase the width of the plot. YiChienLee1010 ***@***.***> schrieb am Fr. 27. Dez. 2024 um 15:20:

…

Hi I want to plot TADs on my matrix but have some problems and I am not sure which step caused the issue. I am using Omni-C data, and I create 256 sets of 4 base random restriction enzyme sites to build a matrix. My commands for each step are as follows. #mapping bwa mem -A1 -B4 -E50 -L0 -t 8 assembly.fa LGH24_R1_v2.fastq.gz > LGH24_R1_v2.sam (same for R2) samtools view -@ 32 -Shb LGH24_R1_v2.sam > LGH24_R2_v2.bam (same for R2) #hicFindRestSite cat random_site.txt | parallel -j 5 'hicFindRestSite --fasta ref.fa --searchPattern {} --outFile cutsites{}.bed' *random_site.txt contain all 256 randome restriction enzyme site #hicBuildMatrix hicBuildMatrix -s LGH24_R1_v2.bam LGH24_R2_v2.bam --outFileName Matrix_bin50000_scaff0001.h5 --QCfolder Matrix_bin50000_scaff0001_QC --restrictionCutFile (list all 256 cutsites) --restrictionSequence (list all 256 randome sequences) --danglingSequence (list all 256 randome sequences) -b Matrix_bin50000_scaff0001.bam --binSize 50000 --threads 8 --region scaff0001 #hicCorrectMatrix hicCorrectMatrix diagnostic_plot --matrix Matrix_bin50000_scaff0001.h5 -o hicCorrectMatrix_bin50000_scaff0001.png hicCorrectMatrix correct --matrix Matrix_bin50000_scaff0001.h5 --filterThreshold -1.5 5 -o hicCorrectMatrix_bin50000_scaff0001.h5 #hicFindTADs hicFindTADs --matrix hicCorrectMatrix_bin50000_scaff0001.h5 --outPrefix hicCorrectMatrix_bin50000_scaff0001_TAD_default --correctForMultipleTesting fdr make_tracks_file --trackFiles hicCorrectMatrix_bin50000_scaff0001.h5 hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains.bed -o scaff0001_TAD_default.ini hicPlotTADs --tracks scaff0001_TAD_default.ini --region scaff0001:1-178105659 -o scaff0001_TAD_default.png scaff0001_TAD_default.ini [x-axis] fontsize = 10 [hicCorrectMatrix_bin50000_scaff0001] file = hicCorrectMatrix_bin50000_scaff0001.h5 depth = 1000 transform = log1p file_type = hic_matrix height = 15 show_masked_bins = false [hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains] file = hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains.bed file_type = domains border_color = black color = none display = triangles title = hicCorrectMatrix_bin50000_scaff0001_TAD_default_domains height = 15 overlay_previous = share-y labels = false fontsize = 10 scaff0001_TAD_default.png scaff0001_TAD_default_hight15.png (view on web) <https://github.com/user-attachments/assets/349c1f69-ad6a-4bc1-a05d-91e3043ba504> I also tried --binSize 10000 at the hicBuildMatrix step, but it also doesn't work. I had set different highs to see any difference, but it still looked weird. I had set depths from 10kb, 40kb, and 120kb, but also no better result. Is this related to bin size? or the plotting parameter? Hicexplorer version 3.7.6 Any suggestions? Thanks! — Reply to this email directly, view it on GitHub <#920>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ADGQCAGHE3V6OJ6XB77O4XT2HVO23AVCNFSM6AAAAABUIWBVP6VHI2DSMVQWIX3LMV43ASLTON2WKOZSG43DAOJUGE3DOMQ> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[YiChienLee1010]

Hi,
Thanks for your reply!!!
I tried increasing the width from 1000 to 10000000. But it still does not have a better look; I guess 170M is too large for making a plot?
Anyway, it looks much better and is expected after shrinking the range to 5MB and increasing the width, as you suggested.

Thank you so much for your time and suggestions.
Best,
YiChien