TruSeq PCR Free LT Kit Issues

[addyblanch]

Hi all would appreciate any input on an issue i'm having.

I've moved across to the TruSeq PCR free kit and seem to be struggling with generating a library.

I'm fragmenting by covaris to 550bp in 50ul RSB as described and I've followed the rest of the protocol to the letter, but my best prep is still only 14pM of ligated adaptors (according to Kapa qPCR quantification). I still have approx 400ng of DNA left (quantified by qubit) which i was expecting due to the size selection steps using the beads.

I've asked tech support and they aren't much help.

I've read that sometimes the adaptors need to be denatured before ligation, Illumina replied with "the protocol has been optimised for the conditions published".

Any ideas would be much appreciated

Adam

[pmiguel]

Hi all would appreciate any input on an issue i'm having.

I've moved across to the TruSeq PCR free kit and seem to be struggling with generating a library.

I'm fragmenting by covaris to 550bp in 50ul RSB as described and I've followed the rest of the protocol to the letter, but my best prep is still only 14pM of ligated adaptors (according to Kapa qPCR quantification). I still have approx 400ng of DNA left (quantified by qubit) which i was expecting due to the size selection steps using the beads.

I've asked tech support and they aren't much help.

I've read that sometimes the adaptors need to be denatured before ligation, Illumina replied with "the protocol has been optimised for the conditions published".

Any ideas would be much appreciated

Adam

What?! If you denature your adaptors prior to use they won't work at all. T4 DNA ligase needs double-stranded substrates for ligation.

If you suspect that your stocks have been denatured at some point, then you want to get new adaptor stocks.

You might be able to re-anneal the adaptors, but I don't like your chances as they have already been diluted to a working concentration that will not favor duplex formation without long hybridization times.

The double-stranded portion of the adaptors is not very long -- 10 or 12 bases, if I recall correctly. So even thawing the adapter tube with your hands might be enough to melt them apart.

I always had a suspicion that the old TruSeq PCR kit adapters had some thermo-labile chemistry covalently linking the two strands of the Y-adapters together. Hence their insistence that at least one cycle of amplification be done to "break the fork" prior to clustering.

If that was the case, it will not be with the no-amp kits as no pre-clustering amplification is done.

So if you or anyone else in your lab are used to melting your adapter tubes in a heat block or something, that might be your issue. You really want to thaw on ice.

Past that, I always suspect contaminants in the DNA prep inhibiting the ligation or end polishing reactions. We always do an Ampure post-fragmentation to mitigate this issue. But even that doesn't seem like enough. We now try to get our customers to do a final RNAse treatment on their pure DNA and follow up with a Zymo Genomic DNA Clean & Concentrator column.

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Phillip

[addyblanch]

Hi Phillip,

I haven't denatured the adaptors, I had read in a troubleshooting guide that a 70 hold was needed to ensure no secondary artefacts are present. I had miss understood and this is actually part of the thermocycler ligation program. So no damage done

Illumina have replaced the kit however so I have fresh reagents.

The new PCR free kit comes with a Ligation Mix which is kept at -20, the adaptors were handled following the protocol, which states RT thaw. They were then used straight away, but will now try thawing on ice.

I also suspect a contaminant in the DNA, and as or DNA is normally prepared in to TE buffer I have done a phenol DNA prep and resuspended an ethonol washed DNA pellet in the Illumina buffer with RNAase A, fragmented, ampure xp clean up and resuspended in Illumina RSB buffer, but it still failled.

I'll have a look into the Zymo kit thanks. Would you do this and the RNAse step post fragment?

Thanks for the input
Adam

[pmiguel]

Hi Phillip,

[...]

I also suspect a contaminant in the DNA, and as or DNA is normally prepared in to TE buffer I have done a phenol DNA prep and resuspended an ethonol washed DNA pellet in the Illumina buffer with RNAase A, fragmented, ampure xp clean up and resuspended in Illumina RSB buffer, but it still failled.

I'll have a look into the Zymo kit thanks. Would you do this and the RNAse step post fragment?

Thanks for the input
Adam

We try to get the customer to do it before bringing us the sample. RNAse first, to degrade RNA small enough that the genomic DNA purification column will exclude the degraded RNA oligos. Then the column -- hopefully removing any last traces of small molecule contaminants (eg, phenol, maybe even cesium if you are getting DNA from an "old school" researcher) or even DNAses. Then we would sonicate.

Then we still do the Ampure prior to end polishing.

One thing to note here is that the pre-clustering PCR amplification gave you a chance to remove from consideration "damaged" amplicons. This would include amplicons with inserts containing non-amplifiable sections. (Due to abasic sites, pyrimidine dimers, etc.)

So if you think about that it becomes a little scary. Before you had this pre-clustering PCR step to shield clustering from weird epigenetic modifications of the source DNA. How robust is clustering to these modifications? If it is more robust, then we are better off than we were before. If it is worse, then there could be issues. Worse, the issues would be sample-specific.

For us it has been "so far, so good". We were getting good results with the pre-amp kit and then seemed to get good results with the no-amp kit. Not a big problem, really. If you decide you really want the amp kit, you can just buy the nano kit. Or the old TruSeq DNA kit until December.

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Phillip

[bioteksampath]

Hi All,

I'm facing this issue in 2018, I just wonder if anyone could provide some suggestions on how to solve this issue. My qPCR shows 100 times less than Qubit concentration. Even I tied again after cleanup with Zymo clean and concentrator kit (elution buffer has very less EDTA 0.1mM). But still the same result!

Thanks in advance!
sam

[kgoglin]

We notice the same thing. Our qPCR concentrations are much less than Bioanalyzer concentrations.

I think the ligation efficiency of the adapters is low.

Have you had any progress on this issue since April?

[pmiguel]

We notice the same thing. Our qPCR concentrations are much less than Bioanalyzer concentrations.

I think the ligation efficiency of the adapters is low.

Have you had any progress on this issue since April?

Impossible to guess what "much less" means in this context. The previous poster mentioned 100x less -- that is clearly an issue.

Anyway, you will want to contact Illumina technical support. That is what they are there for.

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Phillip