ATACseq - large nucleosome peaks

pennypeverell · 03-16-2018, 10:32 AM

Hello everyone!

I am new to ATAC seq and I'm having problems with my libraries. I'd very very thankful if anyone of you is willing to help.

From the bioanalyzer profile I se the 180 bp peak and it should be the mono-nucleosome peak. And that's ok.

The problem is I see other larger peaks (possibly the multiple nucleosomes?) but they have higher intensity than the 180 bp peaks!

I'm a completely noob, but I've been told this is a problem. That larger nucleosomes peaks should be present, but with less intensity compared to the 180 bp peaks. Do you suggest me to sequence these library or throw them away and trying again? Maybe trying to improve my tagmentation step?

I read some similar thread in the forum but nobody is actually having my same bioanalyzer profile (or at least I didn't found it).

I'm attaching some pics of the byoanalizer profiles so maybe you could better figure it out what I am talking about.

Please, any opinion/advice/suggestion would be a great help!

Thanks

Penny

https://ibb.co/iGaZqH

https://ibb.co/naf0VH

Grg91 · 12-11-2018, 12:54 AM

Hi! Did you solve this?

atpoint · 12-11-2018, 08:34 AM

The two bioanalyzer pictures from OP actually look OK to me, clear nucleosomal periodicity. Note that the peak at 180bp is not the mono-nucleosomal but the nucleosome-free peak. From the 180bp, you have to subtract about 100bp for adapter content, leaving you with 80bp inserts which is from nucleosome-free DNA. Fragments of about 200bp are mono-nucleosomal ones.

@Grg91 Can you post a picture of your libraries, so that we can have a look.

Grg91 · 12-12-2018, 12:59 AM

here it is.. what do you think?

atpoint · 12-12-2018, 01:03 AM

Hmm, you have a peak as expected at 180bp and something that can be somekind of nucleosomal peak, but it indeed looks strange. Clearly not as expected. We did ATAC-seq in quiet a number of different cell (lines) and I can say that it typically comes down to the quality of the cells. Did you have excessive numbers of death cells? Did you do any modifications to the standard workflow?

Grg91 · 12-12-2018, 01:21 AM

Would you say that it is over tagmented? I did tagmentation at 55°C for 15 minutes, and I am starting from a pellet of worm nuclei

atpoint · 12-12-2018, 03:26 AM

Yes, this is most likely overtagmentated. It is also too much heat. ATAC-seq works at 37°C for 30' with larger genomes like human and mouse. Worm genomes are much smaller, and therefore you might need to optimize the time and enzyme amount for it. Still, do not go for 55°C. The idea is to leave the chromatin at a native state, and 55°C is not compatible with that plus the transposase is too active. Maybe take a published protocol for Drosophila to get inspiration. I guess the worm genome and Drosophila genomes are more compatible in size than worm and human/mouse.

Grg91 · 12-12-2018, 03:56 AM

Ok thanks! I have started today with a new trial: 37°C incubation and less enzyme.. I'll let you know how it worked!

María Camila Fetiva · 03-01-2019, 06:50 AM

Hello, I performed ATACseq preparation of librarys I used 50.000 cells counted by en bauer chamber, and following the protocol of Buen rostro, I did 25 cycles in total of PCR. Can someone tell me if this library looks ok? (attached pdf 1st sample) I am not sure about it. Thank you so much for your help

Best

Camila

firsal · 08-07-2019, 09:17 PM

Hi,

I have run ATAC-seq library preparation for the first time. I need advice about my Bioanalyser result. It seems the mono-nucleosomal ones are not in high concentration compared to multiple nucleosomes. I used 50,000 human cell line. I also followed the OMNI-ATAC-seq protocol and did amplification for 8 cycles.

What optimizations I should do to get the optimal result. The DNA size is based on second and 60 sec relates to 200 bp.

I really appreciate your feedback and comments.

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03-16-2018, 10:32 AM	#1
pennypeverell Junior Member Location: Spain Join Date: Mar 2018 Posts: 1	ATACseq - large nucleosome peaks Hello everyone! I am new to ATAC seq and I'm having problems with my libraries. I'd very very thankful if anyone of you is willing to help. From the bioanalyzer profile I se the 180 bp peak and it should be the mono-nucleosome peak. And that's ok. The problem is I see other larger peaks (possibly the multiple nucleosomes?) but they have higher intensity than the 180 bp peaks! I'm a completely noob, but I've been told this is a problem. That larger nucleosomes peaks should be present, but with less intensity compared to the 180 bp peaks. Do you suggest me to sequence these library or throw them away and trying again? Maybe trying to improve my tagmentation step? I read some similar thread in the forum but nobody is actually having my same bioanalyzer profile (or at least I didn't found it). I'm attaching some pics of the byoanalizer profiles so maybe you could better figure it out what I am talking about. Please, any opinion/advice/suggestion would be a great help! Thanks Penny https://ibb.co/iGaZqH https://ibb.co/naf0VH Last edited by pennypeverell; 03-16-2018 at 10:38 AM.

12-11-2018, 12:54 AM	#2
Grg91 Junior Member Location: switzerland Join Date: Dec 2018 Posts: 5	same profile Hi! Did you solve this?

12-11-2018, 08:34 AM	#3
atpoint Member Location: Muenster Join Date: Apr 2016 Posts: 14	The two bioanalyzer pictures from OP actually look OK to me, clear nucleosomal periodicity. Note that the peak at 180bp is not the mono-nucleosomal but the nucleosome-free peak. From the 180bp, you have to subtract about 100bp for adapter content, leaving you with 80bp inserts which is from nucleosome-free DNA. Fragments of about 200bp are mono-nucleosomal ones. @Grg91 Can you post a picture of your libraries, so that we can have a look.

12-12-2018, 01:03 AM	#5
atpoint Member Location: Muenster Join Date: Apr 2016 Posts: 14	Hmm, you have a peak as expected at 180bp and something that can be somekind of nucleosomal peak, but it indeed looks strange. Clearly not as expected. We did ATAC-seq in quiet a number of different cell (lines) and I can say that it typically comes down to the quality of the cells. Did you have excessive numbers of death cells? Did you do any modifications to the standard workflow?

12-12-2018, 01:21 AM	#6
Grg91 Junior Member Location: switzerland Join Date: Dec 2018 Posts: 5	Would you say that it is over tagmented? I did tagmentation at 55°C for 15 minutes, and I am starting from a pellet of worm nuclei

12-12-2018, 03:26 AM	#7
atpoint Member Location: Muenster Join Date: Apr 2016 Posts: 14	Yes, this is most likely overtagmentated. It is also too much heat. ATAC-seq works at 37°C for 30' with larger genomes like human and mouse. Worm genomes are much smaller, and therefore you might need to optimize the time and enzyme amount for it. Still, do not go for 55°C. The idea is to leave the chromatin at a native state, and 55°C is not compatible with that plus the transposase is too active. Maybe take a published protocol for Drosophila to get inspiration. I guess the worm genome and Drosophila genomes are more compatible in size than worm and human/mouse.

12-12-2018, 03:56 AM	#8
Grg91 Junior Member Location: switzerland Join Date: Dec 2018 Posts: 5	Ok thanks! I have started today with a new trial: 37°C incubation and less enzyme.. I'll let you know how it worked!