Friday, 21 October 2016

Controlling for bisulfite conversion efficiency with a 1% Lamda spike-in

The use of DNA methylation analysis by NGS has become a standard tool in many labs. In a project design discussion we had today somebody mentioned the use of a control for bisulfite conversion efficiency that I'd missed, as its such a simple one I thought I'd briefly mention it here. In their PLoS Genet 2013 paper, Shirane et al from Kyushu University spiked-in unmethylated lambda phage DNA (Promega) to control for, and check, the C/T conversion rate was greater than 99%.






The bisulfite conversion of cytosine bases to uracils, by deamination of unmethylated cytosine (as shown above) is the gold standard for methylation analysis.

Users identify the C/T transitions in a comparison of bisufite treated/untreated samples, or by comparing to a known reference. However bisulfite treatment is a harsh biochemical reaction, and can cause large losses in template DNA. As such controlling for and measuring conversion efficiency is important in making conclusions about the methylation data from NGS experiments. As a reminder - Bisulfite does not convert methylated or hydroxy-mehtylated cytosine allowing users to discriminate between non-methylcytosine (C) and methylcytosine (mC) or hydroxymethylated (hmC) cytosine.

We're likely to start using this control if it works well in the project we have just kicked off. In the paper they added 1ng of to 1000 oocytes before performing a PBAT analysis. We'll aim for 1% spike-in, but need to consider how much to add to each sample, and whether Lambda is the right spike-in as we're using an RRBS method or this project. To check the suitability I grabbed the Lambda sequence from Genbank and did an in silico Msp1 digest using WebCutter2.0. I found 330 cut sites - which should be plenty for checking efficiency.

Want to learn more about bisulfite conversion in general? Take a look at Zymo's website, it's an excellent resource.

13 comments:

  1. Very interesting and appreciate the resource.

    ReplyDelete
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    ReplyDelete
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    ReplyDelete
  10. Hi there,I have had herpes for a little over 2 years now, I am almost 36 years old, female, and totally embarrassed when I have an outbreak. I refuse to go to classes and will make up an excuse not to go to work so people cant see what my face looks like. For treatment I use Oregano and Meleleuca essential oil in my water for internal use or dab a small amount with coconut oil on the affected area. I also rub oregano with coconut oil on the back of neck since the nerves that run into my face stem from my spinal cord. But in all this my herpes was not cure abut some days ago i was on Facebook then i see a post of someone who say abut the herbal medicine that he use to cure her herpes and this girl name is Cassie Burgess Pepitone. So i need the herbal medicine so i need to ask her abut the herbal cure and he told me the a man called DR.SANDY did the medicine for her wich she use to cure her herpes so i ask for the email and she gave me the email i email DR.SANDY and tell him everything abut my problem of getting this herpes from someone 2 years and ask if he can cure my body and my face well DR.SANDY told me that he will do a herbal medicine for me and send it with UPS OFFICE and once i get the medicine i will use it for 14 days i Sid okay and gave him what he ask form. in 5 days i get my herbal medicine and use it as he told me. well after all the procedures and remedy given to me by this man few weeks later i started experiencing changes all over me as the Dr assured me that i have cured,after some time i went to my doctor to confirmed if i have be finally healed behold it was TRUE, so friend if you have this illness i will ask you to email DR.SANDY and tell him abut your problem okay. Email. dr.sandyspelltemple@gmail.com
    dr.sandyspelltemple@hotmail.com
    dr.sandyspelltemple@yahoo.com OR call Whats App Number +2349031726182
    or facebook posg
    https://www.facebook.com/Drsandy-SPELL-HOME-2-226270587825982/

    i do hope this helps someone suffering...I started practicing yoga one year ago and I can say that breathing exercises have helped wonders. Don't sweat the small stuff.
    THANKS DR.SAND FOR WHAT YOU DID FOR ME.
    Be strong!!!!!!!!!

    ReplyDelete
  11. HOW I WAS CURE FROM HERPES WITHIN 8 DAYS WITH THE HELP OF DR MAZIK
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    ReplyDelete
  12. I tried to donate blood for the first time in my life about 4 years ago, and the local county health department telephoned me that afternoon and said I needed to go to see a doctor immediately because I had hepatitis B. I had absolutely no symptoms. I had several blood tests over the course of a year or so, and my doctor said I was a chronic carrier. My sister had Down's syndrome, and my doctor said I probably got it from her as she lived in an institution for a period of time. As the disease progressed all medication stopped working, i was introduced to Health herbal clinic in Johannesburg who have successful herbal treatment to hepatitis B. I spoke to few people who used the treatment here in USA and they all gave a positive response, so i immediately purchased the hepatitis B herbal formula and commenced usage, i used the herbal supplement for only 7 weeks, all symptoms gradually faded away, herbs are truly gift from God. contact this herbal clinic via their email healthherbalclinic @ gmail. com or visit www. healthherbalclinic. weebly. com

    ReplyDelete
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    ReplyDelete