Tuesday, 28 April 2015

Troubleshooting DNA ligation in NGS library prep

We're currently testing some new methods in the lab to find an optimal exome library prep, not the capture just the prep. The ideal would be a PCR-free exome, however we want to work with limited material and so maximising library prep efficiency is key, and we'll still use some PCR. The two main factors we're considering are ligation temperature/time, and DNA:adapter molar ratio. The major impact of increasing ligation efficiency is to maximise library diversity, and this applies whatever your DNA input. Even if you're not working with low-input samples, high-diversity libraries minimise the sequencing required for almost all applications.

During discussions with some users it became evident that not everyone knows what the critical bits of a DNA ligation reaction are and since adpater ligation is key to the success of many NGS library preps I thought it would be worthwhile summarising some key points here.

Image from taken from Bob Lehman's 1974 Science paper

Temperature: DNA ligation is usually performed at 16C, a couple of people I spoke to assumed that this was the optimal temperature for enzyme activity; it is not. Most experiments will use T4 DNA which ligase has optimal activity at a relatively high temperature e.g. 25C for NEB T4 ligase. However high temperature means DNA molecules are moving rapidly and therefore have little opportunity to pair with each other, and hydrogen bonding between the overhanging bases is disrupted. 16C is the most common compromise temperature for high enzyme activity and reasonable molecular kinetics: the chances of two molecules colliding and sticking to each other. To maximise ligation you can drop temperature to 4C for the very best kinetics, but you'll almost certainly need to extend ligation time to an over-night incubation and/or increase enzyme concentrations (and possibly push the DNA molecules closer together by the same PEG-induced molecular crowding as SPRI). An over-night incubation is reasonably convenient for most of us, but not great if you want to sell a kit that can make libraries in one working day!

DNA:adapter molar ratio: In the past we favoured reducing the amount of adapter by diluting those provided in kits. We had seen a noticeable drop in the adapter-dimer band on agarose gels when we did this, and assumed that it created a "cleaner" library. More recent experiments suggest that high adapter concentrations produce more diverse libraries, and that bead-cleanups remove almost all adapter-dimer contamination anyway. The more adapter you add the more likely you are to ligate adapters to all your DNA fragments.

It may be worth testing different molar ratio of DNA to adapter from 1:10 to 1:100 or even higher. 

Other things to consider:
  1. Use clean DNA, you don't want EDTA inhibiting reactions so use low TE buffer or resuspend in nuclease-free water.
  2. Make sure you've remembered to do the end repair of your fragmented genomic DNA to create the correct structure and chemistry for adapter ligation.
  3. ATP drives the ligation reaction but it is degraded by multiple freeze-thaw cycles; use the whole kit or aliquot/replace the buffer for future use.
  4. Consider using ultra-pure ligases, most reputable ligases will be pretty good but any contamination by other enzymes can modify the end structure of the DNA and inhibit ligation.
  5. Add PEG to increase ligation efficiency by molecular crowding.
The ligase reaction explained:  This explanation is taken from Bob Lehman's (Stanford University School of Medicine) 1974 Science paper, and the BiteSizeBio guide (see references below) - the fragmented genomic DNA is end repaired to produce the 5'-phosphate and A-tail required for adapter ligation. The DNA ligase does its job joining these together in three steps.

1) First the enzyme reacts with ATP (see tip 3 above) producing ligase-adenylate (a covalent enzyme-AMP complex where the adenylyl group is joined to the F-amino group of a lysine residue at the active site by a phosphorus-nitrogen (phosphoamide) linkage), which creates the free energy for hydrolysis.

2) Next the adenylyl group is transferred from the ligase to the 5'-phosphate of the fragmented genomic DNA to form a new 5' pyrophosphate linkage.

3) Finally nucleophilic attack of the AMP by the 3'-OH of the T-overhang in the Adapter forms the new phosphodiester bond in the DNA backbone joining the Adapter:DNA molecules together.

A note on the storage of enzymes: What do you do if you forgot your kit out on the bench over-lunch, or even over-night; chuck it in the bin? Not necessarily! Many enzymes are actually quite stable at fridge or even room temperature. After reading an article in BioTechniques back in 2000: Extended stability of restriction enzymes at ambient temperatures, I made up plates for AFLP mapping with lyophilised restriction-ligation mix. Even after many weeks room temperature storage the plates worked pretty well, and for a high-throughput mapping project this saved huge amounts of time. In the paper the group tested 23 restriction enzymes held at room temp for one week and found no loss in activity; three enzymes were tested for long-term storage at room-temp or 4C and showed no loss in activity over 12 months. Enzyme activity even remained after 12 weeks at 37C. To demonstrate this they posted enzymes to a lab in Africa by snail-mail, which retained activity after three weeks in transit.

If you are inspired to test this with a kit you left out last weekend and on precious samples that can never be rerun on your own head be it!

References and resources for DNA ligation troubleshooting: NEB have very good troubleshooting pages including one on ligases, and one for cloning, BiteSizeBio have posts on (five & three more) things to consider before ligation, and on troubleshooting DNA ligation problems. They also recently published a ebook: 10 Things Every Molecular Biologist Should Know that has a chapter on DNA ligation.

Lehman IR (Science, November 1974). "DNA ligase: structure, mechanism, and function".


  1. Nice post! Have you tried exploring different buffer compositions? The word on the grapevine is that it's the buffer that's responsible for the high yields in the Kapa library prep kits.

  2. I've always been very amused by how people pay through the nose for "quick ligase" kits when even the manufacturer's website will tell you it's just regular ligase with PEG in the buffer.

    1. Certain ligases are more efficient than the others. There's no such thing as just a "regular" ligase.

  3. The link to the bitesizebio e-book is no good!

    1. Hmm, works for me, not sure what's going on there.

    2. Sorry. Should've been clearer. It goes to bitesizebio but no content!

    3. I have no trouble. It opens a PDF.

  4. Unless you're preparing dozens of libraries (and even then you can often get a quote for a great price), it's often best to send the material off to service labs (like our own Eurofins Genomics library prep service). Labs like these often have proprietary methods developed over many projects and several years that increase the desired output.