No data can be extract from .hic file whether normalized or not

[YN HAO]

Hi,

Thanks for the excellent tool! We merged the fastq file of two samples to run hicpro, got  ~433M valid interactions and converted  the valid pairs to .hic file. But we can not extract any data for all chromosome from the .hic file and no loops calling at all resolution. However, if we don't merge fastq and analyzed the two samples respectively, dump and HiCCUPS work well.  We are confused whether too large data can cause this problem.

Many many thanks!

YN

[Neva Durand]

Hello,

No, more data would not cause this issue, more like the opposite (and we routinely run experiments with > 1B Hi-C contacts). 

I am not sure what you're trying to do and what is not working, can you be more specific as to your commands, your system, etc? From what you've said it sounds like your hic file is corrupt for the combined samples.

Finally, usually with replicates we do not run from initial fastqs but instead combine after the dedupping step. We would run Juicer on each sample separately with the -e (early exit) flag and then combine via the mega.sh script.

Hope that helps

Neva

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Neva Cherniavsky Durand, Ph.D. | she, her, hers

Assistant Professor |  Molecular and Human Genetics

Aiden Lab | Baylor College of Medicine

www.aidenlab.org

[YN HAO]

Hi Neva,

Thank you for your reply. We get the valid interactions file from Hi-C Pro pipeline with parallel mode on the SLURM and use the CPU-HiCCUPS with Juicer Tools Version 1.22.01 

1. We use the following command to convert the validpairs to hicfile. The 2221-1.allValidPairs is the merged interactions file(~433M)

 /home/software/HiC-Pro/HiC-Pro_3.0.0/bin/utils/hicpro2juicebox.sh \

  -i /home/HiC/allmerge/2221-1_hicpro/hic_results/data/2221-1/2221-1.allValidPairs \

 -g /home/software/HiC-Pro/HiC-Pro_3.0.0/annotation/chrom_mm10.sizes \

 -j /home/software/juicer/juicer/scripts/common/juicer_tools.jar \

 -r /home/HiC/allmerge/mm10_mboi.bed \

 -o ./

2. The loops calling command

 java -jar /home/software/juicer/juicer/scripts/common/juicer_tools.jar hiccups --cpu -m 512 -r 5000,10000,25000 -k KR -f .1,.1,.1 -p 4,2,1 -i 7,5,3 -t 0.02,1.5,1.75,2 -d 20000,20000,50000 2221-1.allValidPairs.hic all_hiccups_loops

We extracted the first 10000000 lines from the 2221-1.allValidPairs file to create hicfile and this small test hicfile works well with Dump and HiCCUPS. We are also trying to select 10000000 lines randomly to test.

Regards with thanks

YN

[Neva Durand]

I would try to load your hic file in Juicebox to be sure it's valid. You don't post the errors you get but it sounded like a corruption issue if dump isn't working. OTOH if dump is just returning nothing, you might be calling it wrong.

 If you have two files of contacts, you should be able to concatenate them to get the combined hic file.  However we can't really speak to other people's software

To view this discussion on the web visit https://groups.google.com/d/msgid/3d-genomics/e05df170-1e48-4710-a16c-c0b7827e4f73n%40googlegroups.com.

[YN HAO]

Hi Neva,

Thanks for your help and so powerful tool.  When we converted the valid pairs to hicfile, no errors or warnings to give out. 

But in the hicpro2juicebox.sh, for the following commands,

java -Xmx120g -jar ${JUICERJAR} pre -f ${TEMP}/$$_resfrag.juicebox ${TEMP}/$$_allValidPairs.pre_juicebox_sorted ${OUTPUTFILE} ${GSIZE}

We set the Xmx to 60G for the first time, we changed this and set 120G to run the hicpro2juicebox.sh again to generate hicfile. At this time, Dump and HiCCUPs seem to  work well. we are waiting for the result.

Regards with thanks,

YN