RRBS Library 4 peaks

[Andersen]

Dear everyone

I have just prepared my multiplexed reduced representation bisulfite sequencing library. It seems like I have 4 peaks on my bianalyzer results arround 180, 235, 300 and 360 base pairs.

Have any of you experienced these peaks before?

I do multiplexed RRBS with Msp1 digestion, TruSeq Nano adapter ligation and cleanup with dynabeads AMPure XS.

Myself Im thinking that it could be that the Msp1 has a high cutting efficiency at these peaks. Do you have any suggestions?

Do you think that these libraries is good to go for sequencing?

http://imgur.com/ODOuuMu

Kind regards from Emil.

[nucacidhunter]

Few points:
1- HS Chip is overloaded. To see real profile Chip should be loaded within specification, otherwise profile will be distorded
2- Is this from human DNA?
3- What was input and how many PCR cycles were done?
4- Peaks are most likely fom microsattelite fragments that contain MspI site
5- Profile looks a bit different from usual but could be due to overloading or overcycling

[Andersen]

Hi Nucacidhunter.

1 - I will try and run a new chip when we are running a full load.
2 - It is from human stromal vascular fraction
3 - Input was 55 ng DNA originally from 12 samples pooled in one library. I did 20 cycles.
4 - Thanks, I will try and look into the microsattelite fragments. I am quite new to sequencing and library prep so had not heard about it before.
5 - Good, sounds like I will need to run another bioanalyzer before going to sequencing.

Thank you for your help!