This is a small experiment, but I compared standard end repair (ER) conditions to a few other ways. My concern was that T4 polymerase concentration is too high for 500ng of sheared DNA which is a standard input that I use...thus leading to poor blunting. So I titrated the units of enzyme a few different ways while holding PNK concentration constant (the same concentration in the standard protocol ER reaction). I also tried to do fill and phosphorylation separately. I also tried to use 0.5U Klenow to replace T4 polymerase to see how it might do. Interestingly, out of all the conditions, the standard recommended protocol worked the best. I measure library quality: (concentration of completely ligated fragments as measured by QPCR/total concentration of DNA yield) X100. This is one way we can assess the quality of our ligations from the library construction process. As shown by the slide, the standard ER condition as recommended by NEBNext modules and Illumina appears to be the best. I am guessing they have tested this extensively, but I had to be sure! I am also told by many others who look at ligation efficiency for next gen libraries that 20-25% ligation is actually really good. I have other slides I can share if you are interested showing library quality across >300 samples. Although I haven't tested side by side, slowly annealed adapter seems to make a difference. Overall, I believe the first 2 steps of library construction are not very efficient. I have another slide here where I tried to "remake" 3 libraries that were poorly ligated. This slide shows that going all the way back and remaking the library again made the biggest difference. So I haven't done a direct comparison between Klenow and T4 polymerase at the same concentration, which is what you originally asked, but I might predict that there is probably no advantage to either (one needs to use 12U of either enzyme with 40U PNK in the ER step). I am guessing there is a simple reason why Klenow has been omitted from commercially available ER enzyme mixes. Sorry this is so long.

It might has another blend of enzyme. In addition, each provider has optimised the best condition for the enzyme and the buffer.

My best guess it is a compromise of all three enzymes' ideal reaction temperatures. NEB recommends using T4 DNA polymerase at 12 C, Klenow at 25 C, and PNK at 37 C. So I am guessing 20 degrees is middle of the road. The problem is that I have some data showing that end repair is not very efficient with this mix and wondering if I should split this step into 2 to see if my ligation efficiency improves. I have recently looked at over 500 samples and the ligation efficiency at best is 5%. I tested other adapters and get the same result. The only company I have found that has really test this thoroughly so far is KAPA Biosystems. Amr if you have any recommendations for improving ligation efficiency let me know!

Hi Kenneth,

Can I ask what is the sequencing platform are you using? Are you trying to make the library yourself? What did you use for DNA shearing? What are the set of adaptors you used? You mentioned you used different adaptors.

Thanks.

Illumina sequencing. Make libraries ourselves with NEB Next modules. We use Covaris for shearing. We get oligos from a local company and from IDT and anneal ourselves. I compared different adapters from different places and a neighboring genome services lab. I was suspicious that my adapters were bad, but this doesn't seem to be the case! KAPA tells me that a 20% ligation efficiency is good and that a "normal" efficiency is 10%. This sounds pretty bad to me!

Well, this sounds is very interesting. I am using Ion Torrent and some people try to substitute the DNA ligase of Life Technologies kit by the T4 ligase from Thermoscintific and it worked well with them.

Good luck with that.

This is a small experiment, but I compared standard end repair (ER) conditions to a few other ways. My concern was that T4 polymerase concentration is too high for 500ng of sheared DNA which is a standard input that I use...thus leading to poor blunting. So I titrated the units of enzyme a few different ways while holding PNK concentration constant (the same concentration in the standard protocol ER reaction). I also tried to do fill and phosphorylation separately. I also tried to use 0.5U Klenow to replace T4 polymerase to see how it might do. Interestingly, out of all the conditions, the standard recommended protocol worked the best. I measure library quality: (concentration of completely ligated fragments as measured by QPCR/total concentration of DNA yield) X100. This is one way we can assess the quality of our ligations from the library construction process. As shown by the slide, the standard ER condition as recommended by NEBNext modules and Illumina appears to be the best. I am guessing they have tested this extensively, but I had to be sure! I am also told by many others who look at ligation efficiency for next gen libraries that 20-25% ligation is actually really good. I have other slides I can share if you are interested showing library quality across >300 samples. Although I haven't tested side by side, slowly annealed adapter seems to make a difference. Overall, I believe the first 2 steps of library construction are not very efficient. I have another slide here where I tried to "remake" 3 libraries that were poorly ligated. This slide shows that going all the way back and remaking the library again made the biggest difference. So I haven't done a direct comparison between Klenow and T4 polymerase at the same concentration, which is what you originally asked, but I might predict that there is probably no advantage to either (one needs to use 12U of either enzyme with 40U PNK in the ER step). I am guessing there is a simple reason why Klenow has been omitted from commercially available ER enzyme mixes. Sorry this is so long.