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Old 10-30-2016, 11:05 AM   #1
Andersen
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Location: Copenhagen

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Default 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
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Old 10-31-2016, 01:28 AM   #2
nucacidhunter
Jafar Jabbari
 
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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.
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Old 10-31-2016, 08:18 AM   #3
jdk787
josh kinman
 
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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.
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Old 11-02-2016, 02:04 AM   #4
Andersen
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Thank you very much both of you. The samples have now been sequenced. And I will test the analysis in not too long.
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