Out of 70041 loci 70041 were discarded with biases not in range [0.5 2]

[nisar-BIGPM]

Hi,
Thank you for such a nice tool for Hi-C data processing!
I am processing DNase-HiC data. I want to apply Fithic2 to my data; I have prepared the input files from HiC-Pro.

For example:
-i

-f

-t

I proceed to fithic with these input files; however, I encounter error #42. After, reading all comments about #42 and #39, I have removed the biases using: awk '{if ($3 > "0.5" && $3 < "2"){ print}}' and from fragments file I've removed all the validpairs with 0 using: awk '{if ($4 > "0" && $5 > "0"){print }}'.

After, that now I have
-t

-f

After this trimming these files, I used this command to run Fithic:

• fithic -i fithic.interactionCounts.gz -f fragmentMappability.gz -t biases.gz -r 10000 -o ./fithic-out3/

After Fithic2 ran successfully on my files, when I checked the log file, I noticed that " Out of 70041 loci 70041 were discarded with biases not in range [0.5 2]".

Here, is the log file:

And my significance file has all the values are equal to zero and this is the significance file:

I am wondering how to resolve this issue. I don't know where I am making mistakes. I really appreciate it if you have a look into this and help me to fix this problem.

Thank you!
Best,
Nisar

[ay-lab]

There is a problem with having “” around your numbers in awk. Use 0.5 or 2, not “0.5” or “2”. However. That’s not the reason why fithic doesn’t run, the actual problem is that your bias values are very weird and not centered on 1 as they should be.

On Fri, Jul 15, 2022 at 17:06 Nisar Ahmed ***@***.***> wrote: Hi, Thank you for such a nice tool for Hi-C data processing! I am processing DNase-HiC data. I want to apply Fithic2 to my data; I have prepared the input files from HiC-Pro. For example: -i [image: Screen Shot 2022-07-15 at 4 25 27 PM] <https://user-images.githubusercontent.com/108051500/179232277-494d7dca-85d4-4459-aeb3-47468ae2c584.png> -f [image: Screen Shot 2022-07-15 at 4 27 43 PM] <https://user-images.githubusercontent.com/108051500/179232574-147cdb8c-e4b8-419b-8fad-f93fe1ba78a2.png> -t [image: Screen Shot 2022-07-15 at 4 28 24 PM] <https://user-images.githubusercontent.com/108051500/179232686-89933281-a487-421d-8d03-e4f31eb1fe74.png> I proceed to fithic with these input files; however, I encounter error #42 <#42>. After, reading all comments about #42 <#42> and #39 <#39>, I have removed the biases using: awk '{if ($3 > "0.5" && $3 < "2"){ print}}' and from fragments file I've removed all the validpairs with 0 using: awk '{if ($4 > "0" && $5 > "0"){print }}'. After, that now I have -t [image: Screen Shot 2022-07-15 at 4 38 14 PM] <https://user-images.githubusercontent.com/108051500/179234416-bc89e5dc-793a-4455-bd5f-50cbfbc3f49e.png> -f [image: Screen Shot 2022-07-15 at 4 37 20 PM] <https://user-images.githubusercontent.com/108051500/179234276-13236400-031b-4847-a342-b869afdfac5a.png> After this trimming these files, I used this command to run Fithic: - fithic -i fithic.interactionCounts.gz -f fragmentMappability.gz -t biases.gz -r 10000 -o ./fithic-out3/ After Fithic2 ran successfully on my files, when I checked the log file, I noticed that " *Out of 70041 loci 70041 were discarded with biases not in range [0.5 2]*". Here, is the log file: [image: Screen Shot 2022-07-15 at 4 54 35 PM] <https://user-images.githubusercontent.com/108051500/179237471-5fccf3db-9021-4dc2-826b-5afc16daad5c.png> And my significance file has all the values are equal to zero and this is the significance file: [image: Screen Shot 2022-07-15 at 4 56 39 PM] <https://user-images.githubusercontent.com/108051500/179237831-d4227e95-44c3-43b3-89a1-5e48c07f2bf9.png> I am wondering how to resolve this issue. I don't know where I am making mistakes. I really appreciate it if you have a look into this and help me to fix this problem. Thank you! Best, Nisar — Reply to this email directly, view it on GitHub <#56>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AEJTNJBB37JDDVQRQ3OIDMDVUFV6NANCNFSM53VWU54A> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

-- -ferhat

[nisar-BIGPM]

Hi,
Thank you for your reply.
I ran Fithic2 without biases file, and it gives me the results:

However, I want to run it with a biases file. Keeping your comment in mind, I checked the biases file and also created new biases file using HiCKRy.py with HiC-Pro interactions and fragments.

I also filtered the biases with awk '{if ($3 > 0){ print}}'| awk '{if ($3 < 2){ print}}' and fragments with awk '{if ($5 > 0){print }}'

I used these files to run fithic2. However, it gives me the same results and all the loci were again discarded.

Could you please tell me how to fix the biases file? I appreciate any suggestions regarding this issue.

I am looking forward!

Many thanks for considering my request.

Best,
Nisar

[ay-lab]

Seems like there is an issue with your normalization. Maybe about its convergence or maybe about how your reads are distributed. Could you plot coverage tracks per each bin across the genome and/or look at a histogram of coverage (reads per bin)? Bias vector should be normally distributed around 1 (with a mean 1) for FitHiC to work reasonably.