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enzymatic shearing of long-range PCR product? archgen Sample Prep / Library Generation 0 10-24-2012 02:35 PM
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Old 05-14-2013, 08:40 AM   #1
JPC
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Default pooling long range PCR

Hello, I have an experiment with 20 over lapping long range PCR fragments that span a single gene. The average size is 5kb (max 10, min 1.5kb) with equimolar pooling the balance between fragments is poor, we have seen this with a number of samples and the pattern is consistent, some fragments have a few hundred fold coverage, others >100,000x (we're aiming for deep coverage over all amplicons)

Using our results from equimolar pooling we increased the proportion of template for weak fragments but the pattern of high/low coverage between fragments is exactly the same?

Is anyone having better results pooling PCR for NGS? and why would one PCR fragment be more successful than another when they are sheared together?! (GC content is not significantly different).

JPC
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Old 05-14-2013, 09:04 AM   #2
SNPsaurus
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Shearing is less efficient for smaller fragments. Do you see a correlation between coverage and amplicon size? How are you carrying out the shearing?

Other than that, your DNA quantitation may not be accurate. How are you determining the yield from the PCR reaction? Qubit? Nanodrop? Plate reader? qPCR?
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Old 05-15-2013, 12:35 AM   #3
JPC
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We shear with the Covaris and check it with the Bioanalyser, we've also checked with agarose gel to see if there is a significant quantity of un-sheared product, on top of that we've seen the same profile with products that were sheared after being concatinated.

PCRs are quantified with the qubit before pooling, we also use the qubit to quantify samples before they are pooled and we don't have any problems there. Have you had better success with pooling lrPCR? it would be good to know that it can be done rather than chasing a lost cause!
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Old 05-15-2013, 07:13 AM   #4
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Can you afford to quantify your samples with qPCR? The Qubit (and the BioAnalyzer) are showing you all the DNA of the right size, regardless of whether the adapter sequences are attached. If you use the KAPA quant kit, you'll only be quantifying DNA that can stick to the flow cell.
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Old 05-15-2013, 08:03 AM   #5
JPC
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Hi microgirl, we are pooling the PCR then prepping it as one library so there are no adapters to check for at the pooling stage. When we pool 2 libraries we get good balance between the samples but it's the PCR fragments within the samples that are uneven in their sequence success.

JC
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Old 05-15-2013, 08:20 AM   #6
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That does sound strange. Particularly if you've pooled the PCR products, looked at the bias, then used the same PCR product to re-normalize and ended up with the same bias. Adding more of known quantities should change something!

I would try Nextera tagmentation to see what that does. Have you tried shearing your PCR products separately just to see how your settings work on each size? I still think the 1.5 kb fragment will shear much less effectively than the 10 kb. But a 1000-fold swing in coverage is a bit much!

Are you sure your PCR products are the right thing? Maybe its a mapping issue if you are mostly getting off-target amplification.
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Old 05-16-2013, 07:50 AM   #7
JPC
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Yes we have tried tagmentation and confirmed the sequences with Sanger, also there is no correlation with product size and success (nor GC content, or even % of repetitive DNA).

While I do appreciate everyones help no one has actually mentioned if they have had success doing this sort of thing themselves? has anyone actually pooled long range PCR for running on the MiSeq?
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Old 05-16-2013, 10:32 AM   #8
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My academic lab has pooled mtDNA amplifications (2 separate amplicons) from multiple samples without any problem on the HiSeq. I don't know if the coverage balance was perfect, but certainly not anything like what you see.
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