SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Very low efficiency adapter ligation with amplicons and PCR free Kurt Lamour Illumina/Solexa 15 10-02-2015 08:18 AM
Ultra-low DNA yield library prep. francicco Sample Prep / Library Generation 16 05-16-2015 07:27 PM
Low ChIP DNA yield for histone ChIPs dv88 Sample Prep / Library Generation 11 04-28-2015 03:39 PM
Low DNA yield in ChIP - possible workarounds? sequencer2012 Sample Prep / Library Generation 6 01-13-2015 01:57 AM
Nextera DNA library - low yield? sweetph3 Sample Prep / Library Generation 9 03-23-2014 03:03 AM

Reply
 
Thread Tools
Old 02-10-2016, 07:17 AM   #1
reprogrammer
Member
 
Location: Los Angeles

Join Date: Jan 2016
Posts: 10
Default Low DNA yield after adapter ligation

Hi guys,

I am generating RRBS library recently. In the whole process I use Beckman Ampure Xp beads to purify (cleanup and elute) DNA. But only the step of adapter ligation showed a extremely low efficiency of DNA yield (~35%).

The efficiencies of End repair and Add A steps are very good (over 90%). Do you have any experience about that? What should I do to improve the efficiency after adapter ligation?

Thank you so much!
reprogrammer is offline   Reply With Quote
Old 02-10-2016, 09:25 AM   #2
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 64
Default

Hello Reprogrammer,
It is going to be hard to answer this question without more information.

Can you describe the protocol that you are using?
Listing your steps and including the sample and bead volumes that you are using for your cleanups would be a good start.
jdk787 is offline   Reply With Quote
Old 02-10-2016, 09:50 AM   #3
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 402
Default

Ligation efficiency will depend a lot on the appropriate adapter concentration. Do you see any adapter dimers after the ligation.
luc is offline   Reply With Quote
Old 02-22-2016, 10:07 AM   #4
reprogrammer
Member
 
Location: Los Angeles

Join Date: Jan 2016
Posts: 10
Default

Quote:
Originally Posted by jdk787 View Post
Hello Reprogrammer,
It is going to be hard to answer this question without more information.

Can you describe the protocol that you are using?
Listing your steps and including the sample and bead volumes that you are using for your cleanups would be a good start.
Hi jdk787,

I summarized my protocol and put it in the attachment. Would you help me take a look is there anything wrong, please?
Thank you so much.
Attached Files
File Type: pdf SOP of DNA methylation.pdf (137.4 KB, 31 views)
reprogrammer is offline   Reply With Quote
Old 02-24-2016, 12:01 PM   #5
reprogrammer
Member
 
Location: Los Angeles

Join Date: Jan 2016
Posts: 10
Default

I noticed that I made a conceptual mistake. What I am doing is whole-genome DNA methylation but not RRBS since I am using sonication biorupter to fragment genomic DNA.
reprogrammer is offline   Reply With Quote
Old 02-26-2016, 08:31 AM   #6
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 64
Default

Quote:
Originally Posted by reprogrammer View Post
Hi jdk787,

I summarized my protocol and put it in the attachment. Would you help me take a look is there anything wrong, please?
Thank you so much.
Hello Reprogrammer,
Sorry for the delayed response, I've been out of town.
One big problem that I see is that you are trying to blunt, phosphorylate, and A-tail your DNA using just Klenow Fragment (3-5 exo)
This enzyme is recommended for A tailing previously blunted and phosphorylated DNA, not blunting and A-tailing.

You need to blunt your DNA first, and then A-tail.

Also, you should check to make sure that your fragmented DNA is in a size range that is compatible with your protocol by either running a portion of it on a gel or BA. If you are over fragmenting your starting material you may be losing it during your cleanups.

For your PCR amplification, I think you can skip the Ampure cleanup after the post conversion column cleanup and just go straight in to PCR.

Finally, you need to make sure that you are using a Uracil literate Taq for your amplification since you will have C -> U conversions in your template.

If you want an easier and more straight forward way to make your libraries, many companies sell kits that will supply you with the protocol and all of reagents that you need to perform WGBS and RRBS library prep. Kapa, Illumina, and Bioo Scientific (where I work) to name a few.

Feel free to PM me if you would like info on the Bioo Scientific kit.
jdk787 is offline   Reply With Quote
Old 03-14-2016, 07:31 AM   #7
reprogrammer
Member
 
Location: Los Angeles

Join Date: Jan 2016
Posts: 10
Default

Quote:
Originally Posted by jdk787 View Post
Hello Reprogrammer,
Sorry for the delayed response, I've been out of town.
One big problem that I see is that you are trying to blunt, phosphorylate, and A-tail your DNA using just Klenow Fragment (3-5 exo)
This enzyme is recommended for A tailing previously blunted and phosphorylated DNA, not blunting and A-tailing.

You need to blunt your DNA first, and then A-tail.

Also, you should check to make sure that your fragmented DNA is in a size range that is compatible with your protocol by either running a portion of it on a gel or BA. If you are over fragmenting your starting material you may be losing it during your cleanups.

For your PCR amplification, I think you can skip the Ampure cleanup after the post conversion column cleanup and just go straight in to PCR.

Finally, you need to make sure that you are using a Uracil literate Taq for your amplification since you will have C -> U conversions in your template.

If you want an easier and more straight forward way to make your libraries, many companies sell kits that will supply you with the protocol and all of reagents that you need to perform WGBS and RRBS library prep. Kapa, Illumina, and Bioo Scientific (where I work) to name a few.

Feel free to PM me if you would like info on the Bioo Scientific kit.
Hi jdk787,

Thank you so much for your suggestion. Finally I got the PCR product with your guidance.

First I changed the Taq enzyme to Cx Pfu, I believe this is the major mistake I made. At the beginning I used the PrimeStat which could not recognize U in the template DNA after bisulfite conversion. Second, I over amplified the template DNA with 40 cycles to make sure the product was exist. I am trying to reduce the PCR cycles to 20~25 now.

For the commercial kit, I am contacting with NuGen company which my friend used pretty well with 25 ng DNA input.
reprogrammer is offline   Reply With Quote
Reply

Tags
library cleanup, ngs, rrbs

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 06:54 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO