HiC-Pro Running Error

[Gang Li]

Hi,

I am trying to run HiC-Pro with HiChIP data. Our Hi-ChIP data is made by micrococcal nuclease digestion using the Dovetail kit. This method is based on Micro-C. So no restriction enzyme is used.

I was told to omit [GENOME_FRAGMENT] and [LIGATION SITE]. However, when running step 1, I met the following error.

HiC-Pro_3.1.0/scripts/mergeSAM.py -q 30 -t -v -f *S2_R1_001_GRCm38.bwt2merged.bam -r *S2_R2_001_GRCm38.bwt2merged.bam -o *S2_001_GRCm38.bwt2pairs.bam

[E::hts_open_format] Failed to open file "*S2_R1_001_GRCm38.bwt2merged.bam" : No such file or directory

In my previous successful run, I think *.bwt2merged.bam is created via a symbolic link from Mapping_combine.log. I do observe the mapping to *.bwt2glob.bam. Should I manually create the link and continue to run it?

Best,

Gang

[nservant]

Hi,

No you have nothing to do manually in theory.

Could you show me the content of the bwt2 folder ? and how do you set the PAIR1_EXT/PAIR2_EXT in the config ?

Thanks 

[Gang Li]

Hi

Thank you for your replay!

Under bwt2, I got two folders for two samples that we have. See the below for one sample as an example:

- K27ac_Hi_ChIP_patWT

— K27ac_Hi_ChIP_patWT_S1_R1_001_GRCm38.mapstat

— K27ac_Hi_ChIP_patWT_S1_R1_002_GRCm38.mapstat

All *.mapstat are empty.

I set:

PAIR1_EXT = _R1

PAIR2_EXT = _R2

My data:

K27ac_Hi_ChIP_patWT_S1_R1_001.fastq.qz

K27ac_Hi_ChIP_patWT_S1_R2_001.fastq.qz

It seem that it does not read the right R2 files. I highlight the name differences.

Should I try?

PAIR1_EXT = _R1_001

PAIR2_EXT = _R2_002

Best,

Gang

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[Gang Li]

Hi,

I tried to rename the files but got the similar error as before.

[E::hts_open_format] [E::hts_open_format] Failed to open file "bowtie_results/bwt2/K27ac_Hi_ChIP_patWT_S1/K27ac_Hi_ChIP_patWT_S1_R2_GRCm38.bwt2merged.bam" : No such file or directoryFailed to open file "bowtie_results/bwt2/K27ac_Hi_ChIP_patWT_S1/K27ac_Hi_ChIP_patWT_S1_R1_GRCm38.bwt2merged.bam" : No such file or directory


Under bwt2, I have two samples.
- K27ac_Hi_ChIP_patWT
— K27ac_Hi_ChIP_patWT_S1_R1_GRCm38.mapstat
— K27ac_Hi_ChIP_patWT_S1_R2_GRCm38.mapstat

All *.mapstat are empty.

I set:
PAIR1_EXT = _R1
PAIR2_EXT = _R2

My data:

K27ac_Hi_ChIP_patWT_S1_R1.fastq.qz
K27ac_Hi_ChIP_patWT_S1_R2.fastq.qz

Best,
Gang


> On Jul 29, 2022, at 1:39 PM, nicolas.servant <nicolas...@curie.fr> wrote:
>
> Hi
> Indeed I think that renaming the files with simple name "*R1.fastq/R2.fastq" could be a good idea.
> Let me know if it fixes the issue
> N
>
> -------- Message d'origine --------
> De : Gang Li <frankle...@gmail.com>
> Date : 29/07/2022 22:20 (GMT+01:00)
> À : nservant <nicolas.se...@gmail.com>
> Cc : HiC-Pro <hic...@googlegroups.com>
> Objet : Re: [hic-pro] HiC-Pro Running Error

[Gang Li]

Hi,

Thank you for your efficient responses during the travel! I really appreciate that.

See below for one of the bowtie log:

##HiC-Pro mapping

13733317 reads; of these:

13733317 (100.00%) were unpaired; of these:

4089621 (29.78%) aligned 0 times

7974023 (58.06%) aligned exactly 1 time

1669673 (12.16%) aligned >1 times

70.22% overall alignment rate

 

It looks like the *.bwt2glob.bam files were successfully generated, right?

Under bowtie2glob folder:

— *.bwt2glob.bam

— *.bwt2glob.umap.fastq

Under bwt2 folder:

— *.mapstat (empty)

Best,

Gang

On Jul 29, 2022, at 11:10 PM, nicolas.servant <nicolas...@curie.fr> wrote:

Something went from during the mapping.

Please check the bowtie logs.

Do you have other results in bowtie_results ?

Sorry I'm traveling for one month ... difficult to help you without computer !

N

-------- Message d'origine --------

De : Gang Li <frankle...@gmail.com>

Date : 30/07/2022 06:09 (GMT+01:00)

À : "nicolas.servant" <nicolas...@curie.fr>

Cc : nservant <nicolas.se...@gmail.com>, HiC-Pro <hic...@googlegroups.com>

[Gang Li]

[Gang Li]

Hi Nicolas,

Thanks for your efforts on this! 

I attached two small fastq files (downsampled). Please take a look.

I will also take a look at mapped_2hic_fragments.sh.

Best,

Gang

[Gang Li]

Hi Nicolas,

Have you got the chance to take a look at the downsampled data? I recently tried to run HiCPro on a DNase HiC data but got the exact same error.

Best,

Gang

<K27ac_Hi_ChIP_patWT_S1_R2.50k.fastq.gz>

<K27ac_Hi_ChIP_patWT_S1_R1.50k.fastq.gz>

On Aug 6, 2022, at 12:30 AM, nservant <nicolas...@curie.fr> wrote:

Hi,
Sorry for the delay. The config file looks good.
There is something I do not understand ...
In theory, it should run the DNAse mode as GENOME_FRAGMENT is empty.
But based on your log, it seems that this is not the case, and that it runs the digestion mode ...
You have a comment fore GENOME_FRAGMENT but I guess this is not an issue ...
Maybe you could try to add some trace in the script mapped_2hic_fragments.sh, to try to understand what's going on.
Otherwise, if you can send me small files, I can test it here ...
Sorry
Nicolas

Le 01/08/2022 à 19:48, Gang Li a écrit :

Sure. Please see the attached file.

Best,

Gang

On Aug 1, 2022, at 10:43 AM, nicolas.servant <nicolas...@curie.fr> wrote:

Could you send me your config please ?

Thanks

-------- Message d'origine --------

De : Gang Li <frankle...@gmail.com>

Date : 01/08/2022 19:01 (GMT+01:00)