custom PCR enrichment: how to know if it's good enough?

[pyridine]

Hi, I'm new to next-gen sequencing and I'm trying to generate a TruSeq DNA library for sequencing on a MiSeq. My application requires a custom PCR enrichment step to pull out transposon-chromosome junctions, from which I'm starting from scratch and putting together my own protocol based on other previously published protocols - but it is necessarily different since the adapter sequences keep changing and the transposon is different. Basically, how do you know if the PCR is providing adequate and specific enrichment?

I started with 1 ug of genomic DNA and I am following the standard Illumina TruSeq workflow up to the PCR enrichment step. After gel extraction, two samples that I quantified on a Nanodrop had concentrations of 21.3 ng/uL and 16.8 ng/uL, respectively. This is about 50% yield from the original 1 ug input DNA, so I don't think this is too bad considering the large number of purification steps up to this point.

For the PCR enrichment, I purchased an 80-bp biotinylated transposon-specific primer that has the entire universal adapter sequence as a 5' overhang (bound to cause trouble but no forseeable way around it...), and an indexed adapter specific reverse primer that is much shorter. I'm having a tough time doing a lot of PCR optimization due to the limited amount of sample, but with 1 ng input DNA and an annealing temperature gradient, I see visible smears of approximately equal intensity for all annealing temperatures from 51 to 65C, in about the same size range as for the gel extracted template. So I picked an intermediate annealing temperature (a tad lower than the calculated Tm of the transposon binding region) and ran different cycle numbers using about 1.7 ng/uL of template in the reaction, which is the amount of template I would aim for in the actual library enrichment. Quantification of the products on a Qubit showed 5.4 ng/uL after 15 cycles, jumping to 13.7 ng/uL after 18 cycles, then barely climbing after this with a maximum of 17.3 ng/uL after 25 cycles. Not sure if I trust these numbers, but they seem extremely low for a PCR amplification if true.

So is there any way to know whether this PCR product is any good? It's just smear-in smear-out. I suppose I could generate some artificial DNA fragment to use as a control for PCR optimization, but this could take awhile and would never ideally represent the template. It seems that if I stop at 18 cycles to have optimal enrichment with minimal bias, I will most likely still have enough DNA after more purifications and affinity capture to have at least 10 uL of 10 nM DNA for cluster amplification (about 2.6 ng/uL). Is it worth it to just try it out and proceed with sequencing, or should I take more steps to ensure a solid PCR amplification first?

[TonyBrooks]

A few points that immediately come to mind:

Just because you have 50% yield post gel extraction doesn't mean that all of that is going to be adapter-ligated. No adapter ligation = no enrichment.

You don't need the full universal adapter sequence on your transposon specific primer, just the P5 sequence. You can then use the transposon sequence to prime sequencing (you might need some padding in there to keep the Tm of the read 1 primer > 65C).

If you want to check on your enrichment PCR, a trick I've seen is to add SYBR to the PCR and track the amplification on a qPCR machine (ideally one that plots in real-time).

What concentration of primer are you using in your enrichment? It's possible that after 18 cycles you've exhausted the primers in the reaction and hit the plateau. 17.3ng/µL is a lot of library.

[pyridine]

Thanks a lot for the tips.

Assuming the P5 sequence is the part that's complementary to the flow cell, are you saying I should just use a custom sequencing primer? I guess I was trying to avoid that, but it's definitely an option if this PCR really isn't working out.

Yeah I wanted to just do qPCR but I didn't have any SYBR Green immediately available. I think it might be worth rerunning one reaction if I can find any tomorrow.

So you think that the 17.3 ng/uL is plenty to work with? I guess I'm also just really concerned about the quality of it, and knowing that it's really the desired product amplifying. I guess it probably is if it's a smear centered around 400-bp, but it's hard to be sure.

[pyridine]

Forgot to add the primers are 0.3 uM, shouldn't be limiting but a lot could be bound up as primer dimer junk.

[SNPsaurus]

Are there any known loci that are being amplified? You could check enrichment with a qPCR of one or a few control loci.

[TonyBrooks]

17.3ng of a ~400bp library is around 70nM, so more than enough to sequence.

[pyridine]

I tried what was suggested - to run PCR with a known locus, since I know the location of many transposon/chromosome junctions and already have primers on hand. So this chromosome-specific primer was used together with the 80-bp Tn-specific primer with universal adapter overhang, to see if I could pull out the product of a specific size from both the original library and from one of the whole-library PCR reactions yesterday. There is an extremely weak band of approximately the right size (which is smaller than the PCR product smear using the indexed adapter specific primer) that is only visible using the PCR reaction as template. This is after 35 cycles and about 0.5 ng template DNA.

So it seems that the reaction is working, but is is very inefficient. While the overall yield is several times more DNA than ultimately needed, there is still a gel purification, then affinity capture plus one more PCR purification to go, so I'm expecting to lose another 50-70% - although I should have just enough library DNA at this point to pool two PCR reactions together if necessary. Also about 1/8 of the reads will not have any transposon junction in them due to the low efficiency of enrichment, which I suppose is a tolerable number to throw out assuming there's not a whole lot of other unanticipated junk in there.

Guess I just have to try it, but running up against this concentration limit makes me pretty nervous.