Poor Ligation Efficiencies on illumina Libraries

[mollusc]

I was just wondering what are the ligation efficiencies on illumina libraries for people working on it.I get poor efficiencies on QPCR, ranging from 40-5%, and even worst for no PCR libraries.I calculate my efficiency as mentioned below.
QPCR/Bioanalyzer Conc.*100
I did compare on Taqman as wel as sybrgreen kapa reagents, but din't really had a significant difference.
Please let me know if anybody is having similar problems

[gaster]

me too

I have had the same problem. I am using a no-amp approach and with the two libraries I have tried the ligation efficiency (including recovery efficiency) is less than 1%. I have tried both NEB ligase and Enzymatics Ultra-pure ligase.

Do you see an size shift an obvious size shift in your library post ligation indicating that adaptors have been ligated to the insert?

[niceday]

I would be more interested in the ligation efficiency before PCR.
After PCR the amount of ligated material is much higher because PCR only amplifies up properly ligated library.

qPCR results can be quite a bit less than bioanalyser or qubit results as they show all the DNA and qPCR shows the good library.

Depending on your ligation efficiency and how many cycles of PCR you have done will depend on what % of your library is good.

Does anyone else out there have a feel for their ligation efficiency? We have decided to start logging it as a QC step and I would be interested in what sorts of figures other sites are getting.

[Liting]

We have had the same problem.We can not get enough libraries after PCR .We have confirmed that there is noting wrong with the ligase by ligating marker DNA.We think the problem is because of adaptors.We have tried Illumina and NEB kit.The problem is still there.Does anyone tell us how to solve the problem? Thank you very much.

[Heisman]

We have had the same problem.We can not get enough libraries after PCR .We have confirmed that there is noting wrong with the ligase by ligating marker DNA.We think the problem is because of adaptors.We have tried Illumina and NEB kit.The problem is still there.Does anyone tell us how to solve the problem? Thank you very much.

Do you make sure the adapters are hybridized together prior to using them?

[NextGenSeq]

Also make sure your adapters are synthesized with a phosphotioate bond between the last two 3' bases.

[Liting]

Library preparation

Also make sure your adapters are synthesized with a phosphotioate bond between the last two 3' bases.

Thank you very much.Our adapters work well and don't have any problems.We have got enough libraries by PCR.But the size of DNA were smaller.It's abnormal.I can't find how it happens.We already carried on llibraries preparation for two months.It is unsuccessful.

[epistatic]

We go immediately from dA-tailing to ligation to get the best yields as the 3'A is unstable. I also limit the ligation to 15 minutes and stop with EDTA before going to bead purification to avoid ligating too long.

[pmiguel]

We go immediately from dA-tailing to ligation to get the best yields as the 3'A is unstable. I also limit the ligation to 15 minutes and stop with EDTA before going to bead purification to avoid ligating too long.

Do you have a reference to support that the 3' A is "unstable". I can't think of any reason it would be any more unstable than any other 3' overhang.

--
Phillip

[pmiguel]

Thank you very much.Our adapters work well and don't have any problems.We have got enough libraries by PCR.But the size of DNA were smaller.It's abnormal.I can't find how it happens.We already carried on llibraries preparation for two months.It is unsuccessful.

How much smaller? By what assay did you determine size?

Single-stranded DNA migrates more slowly on several types of Bioanalyzer chips than double-stranded. So single stranded regions (eg, Y-adapters), could cause amplicons to "look" longer than they really are on a Bioanalyzer chip. After "enrichment" PCR the Y-adapters would no longer be single-stranded.

--
Phillip

[Shifty]

I have similar problems with the Truseq adapters on no-amp materiaal.
2,5 uq of DNA sheared with covaris (max peak 400 bp)

end-repair
add 'a
ligation (all in NEB kit) according to protocol and AMPURE xp washes between.
size selection with Agilent XT 400 to 500 bp

On bioanalyzer I get a nice peak (2 nM concentration)

But poor cluster results.

I thought that I could see good results from the test PCR to check ligation. But cluster density shows otherwise. Next week qPCR test.

Never had this problem before. Happend now twice.

[pmiguel]

If you are not doing amplification you might be able to get an approximate assay on % of fragments with adapters on the agilent chip. Just save an aliquot of the fragmented, end polished, A-tailed DNA. Use the rest for ligation as normal. Then run pre- and post- ligation on the Agilent high sensitivity DNA chip.

You may be able to see a shift in the size of the fragments. The adapters comprise a 63 and a 58 bp oligo. So, you would expect at least a 120 bp shift for fragments that have been ligated at both ends to adapters. (The shift might be larger due to ssDNA drag.)

--
Phillip

[sehrrot]

Dear all,

I hope this would be helpful
To increase ligation efficiency,
1. Purify your samples after covaris shearing. I've noticed that some samples had been kept in TE buffer and ligation wasn't worked well. So, every time after shearing, I purify samples using Illumina resuspension buffer then make dilution 20 ng/ul (for 1 ug input)
2. Check your concentration before PCR and then compare the concentration b/w pre-pcr and post-pcr. I've used both gel-free and gel method; PCR efficiency of gel-free method is around 10X and that of gel method is over 30.
3. Check the expiry date of Truseq library prep kit. This is pretty short than the previous version.

[fluo]

sehrrot is right.

check the expriy date of Truseq library prep kit. It is important to avoid expired reagent.
I used expired reagent which was out of expriy date about a month and got a very poor PCR efficiency(about 2X). Now, I changed new reagents and get a PCR efficiency which is over 20X.

[sehrrot]

Hey fluo

Have you ever tried Herculase pol. instead of Phusion HF buffer? I feel it gives me more efficient amplification..