RRBS library long fragments

Andersen · 10-30-2016, 11:05 AM

Dear All,

I just made a RRBS library. The problem is that I have a lot of long fragments that is not really good since they probably do not contain CpG rich areas.

This is the profile of the library:
https://i.imgsafe.org/63616d827a.jpg

Can anyone help me with what I should do?

It is made from 16 samples pooled together. DNA is from blood with good quality. Msp1 digestion worked.

Please help. Do you believe that i can rescue the library with size selection?

Cheers
Emil

nucacidhunter · 10-31-2016, 01:28 AM

The short fragments looks typical of human or mouse RRBS but I think that PCR extension time has been excessive resulting in large fragments amplification as well or PCR has been overcycled. It is OK to sequence it without any size selection because clustering will favour short fragments so a small number of reads will be from large fragments. You might need to increase sequencing depth in comparison to a size selected library for optimum coverage.

If large fragments are result of MspI digestion (not degraded DNA that has been end repaired and adapted) then they will have at least one CpG site. The only issue is that you probably will not get enough sequencing coverage for those.

jdk787 · 10-31-2016, 08:18 AM

The markers for this BA trace are also being called incorrectly, so you will want to go back and manually set them in order to get an accurate size distribution. That upper peak should be at 10380bp, but it is showing up at ~800. This will cause the software to underestimate the fragment sizes.

Andersen · 11-02-2016, 02:04 AM

Thank you very much both of you. The samples have now been sequenced. And I will test the analysis in not too long.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
ultra long fragments capturing	Thuur	Sample Prep / Library Generation	5	06-01-2020 09:41 AM
RRBS NGS library prep	xiefanfang@gmail.com	Illumina/Solexa	4	02-10-2016 03:30 PM
RRBS Library 4 peaks	Andersen	Epigenetics	2	10-28-2015 02:44 AM
RRBS library prep.	majo82	Epigenetics	5	04-01-2011 01:32 AM

10-30-2016, 11:05 AM	#1
Andersen Member Location: Copenhagen Join Date: Oct 2015 Posts: 15	RRBS library long fragments Dear All, I just made a RRBS library. The problem is that I have a lot of long fragments that is not really good since they probably do not contain CpG rich areas. This is the profile of the library: https://i.imgsafe.org/63616d827a.jpg Can anyone help me with what I should do? It is made from 16 samples pooled together. DNA is from blood with good quality. Msp1 digestion worked. Please help. Do you believe that i can rescue the library with size selection? Cheers Emil

10-31-2016, 01:28 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	The short fragments looks typical of human or mouse RRBS but I think that PCR extension time has been excessive resulting in large fragments amplification as well or PCR has been overcycled. It is OK to sequence it without any size selection because clustering will favour short fragments so a small number of reads will be from large fragments. You might need to increase sequencing depth in comparison to a size selected library for optimum coverage. If large fragments are result of MspI digestion (not degraded DNA that has been end repaired and adapted) then they will have at least one CpG site. The only issue is that you probably will not get enough sequencing coverage for those.

10-31-2016, 08:18 AM	#3
jdk787 josh kinman Location: Austin Join Date: Apr 2014 Posts: 71	The markers for this BA trace are also being called incorrectly, so you will want to go back and manually set them in order to get an accurate size distribution. That upper peak should be at 10380bp, but it is showing up at ~800. This will cause the software to underestimate the fragment sizes.

11-02-2016, 02:04 AM	#4
Andersen Member Location: Copenhagen Join Date: Oct 2015 Posts: 15	Thank you very much both of you. The samples have now been sequenced. And I will test the analysis in not too long.