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Old 09-26-2013, 01:41 AM   #1
LAGroh
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Location: Utrecht

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Default Adapter primer design issues, and why phosphorothioate bonds?

Hi, our group is performing a population study of stem niche succession in colorectal crypts; not too interesting for you guys. However this does mean that we will have some 300 unique samples though, so in our library prep we would like to attach barcodes to both the front and the back of our amplicons to reduce the number from 300 unique primers to 30, and since the ligation kits are really expensive we’re planning to also include the A adapter and the P1 sequence into our primers as well. The issue is, we were given sequences from the sequencing department here in the hospital, but they don't seem to match any of the literature. Below are the sequences. P1 B comp appears to be the compliment of P1 A but with the odd addition of “*T*T” at the 3’ end (presumably “in” the DNA amplicon :S ).


P1 A: 5'-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT-3'

P1 B "comp": 5'-ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG*T*T-3’

Lit: 5' -CCTCTCTATGGGCAGTCGGTGAT- 3'

* = phosphorothioate bonds.

Interestingly, the red section is the same sequence as the sequences found in literature. So my question is, which sequence should I use to design my reverse primer (also let me know if I should use the complimentary strand to any of these sequences)?
The P1 A and P1 B sequences seem to be the same sequences used in the KAPA Adapter Kits technical data sheet, which doesn’t provide much information on why things are used and unfortunately I can’t find much other information on this kit otherwise.

Also I read that phosphorothioate bonds prevent the degradation by exonucleases, but I have a hard time figuring out when my DNA will be exposed to exonucleases during the sequenceing procedure. Just to keep an eye on the costs, would it be possible/recommended to do without them?
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Old 09-26-2013, 09:01 AM   #2
jwfoley
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Quote:
since the ligation kits are really expensive we’re planning to also include the A adapter and the P1 sequence into our primers as well.
A certain popular ligation kit is a ridiculous waste of money because it's just a regular ligation kit with PEG added to the buffer, but at any rate, if you can build adapters into your primers and avoid ligation, that's an improvement for non-cost reasons too: ligation reduces your yield and library complexity. (So does any unnecessary step, but especially ligation since it's intermolecular and low-efficiency.)

Quote:
The issue is, we were given sequences from the sequencing department here in the hospital, but they don't seem to match any of the literature.
I don't recognize them either - maybe you could specify what sequencing platform they're for?

Quote:
Also I read that phosphorothioate bonds prevent the degradation by exonucleases, but I have a hard time figuring out when my DNA will be exposed to exonucleases during the sequenceing procedure. Just to keep an eye on the costs, would it be possible/recommended to do without them?
They're there for a reason and the cost is negligible. The exonuclease is actually the 3'->5' exo activity of the polymerase (which is what gives it its fidelity); you don't want your polymerase chewing up your primers instead of synthesizing off them. But it basically can't digest thiolated DNA. Then why do you need more than one phosphorothioate? Because when you synthesize the oligo, you get a racemic mixture of one enantiomer that stops exonucleases and one enantiomer that doesn't. So with 50-50 odds of a useless phosphorothioate at each of two positions, there's only a 25% chance that any given molecule is vulnerable to exonuclease digestion.
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Old 09-26-2013, 11:42 PM   #3
LAGroh
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Originally Posted by jwfoley View Post
I don't recognize them either - maybe you could specify what sequencing platform they're for?
Ahem... Yes I could see why that would be important. We're going to use Ion Torrent PGM.
Sorry for whatever reason I thought I was in the PGM section!

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Originally Posted by jwfoley View Post
They're there for a reason and the cost is negligible. The exonuclease is actually the 3'->5' exo activity of the polymerase (which is what gives it its fidelity); you don't want your polymerase chewing up your primers instead of synthesizing off them. But it basically can't digest thiolated DNA. Then why do you need more than one phosphorothioate? Because when you synthesize the oligo, you get a racemic mixture of one enantiomer that stops exonucleases and one enantiomer that doesn't. So with 50-50 odds of a useless phosphorothioate at each of two positions, there's only a 25% chance that any given molecule is vulnerable to exonuclease digestion.
Ok thanks that makes sense. Strange how I haven't been able to read this anywhere in the manuals :S
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